Methods of prevention and treatment of asthma, and allergic conditions

ABSTRACT

The present invention relates to allergy vaccines and methods of treating and/or preventing asthma, and allergic conditions. The invention is based on the discovery that inhibiting the ligand/receptor interactions involving, e.g., IgE, IL-3, IL-4, IL-5, IL-6, IL-10, IL-13, interferon-alpha, histamine, leukotriene, and their respective receptors, inhibits production of IgE thereby treating or preventing such diseases or conditions. Competitive inhibition of such receptor/ligand interactions is accomplished by immunizing a human or veterinary patient with the interleukin, interferon-alpha, histamine, leukotriene, their receptors, in any combination. Also, the invention relates to inhibiting receptor/ligand interactions involved in IgE production by competitively inhibiting such interactions by administering antibodies to the ligands, receptors, or both, as well as by administering analogs of the receptors (e.g., soluble receptors not associated with a cell).

BACKGROUND OF THE INVENTION

The development of allergy or other types of immune hypersensitivity isan important undesirable effect of acquired immunity in mammals,particularly humans. There are several types of allergy. The presentinvention is concerned with atopic allergy. Atopic allergy ischaracterized by an excess production of IgE antibody, which attaches tomast cells and basophils. A single mast cell or basophil may bind asmany as a half a million molecules of IgE. When an antigen binds to atleast two IgE molecules bound on the surface of a mast cell or abasophil, the cell ruptures or otherwise is caused to release severalsubstances including histamine, leukotrienes (previously referred to as“slow reacting substance of anaphylaxis”), eosinophilic chemotacticfactor, proteases, neutrophil chemotactic substance, heparin, plateletactivating factors and bradykinin. Release of these substances resultsin dilation of local blood vessels, attraction of eosinophils andneutrophils to the reactive site, damage to local tissues by proteases,increased permeability of the capillaries and loss of fluid into thetissues, and contraction of smooth muscle cells. A number of differentabnormal tissue responses ensue including anaphylaxis, urticaria, hayfever and asthma.

In anaphylaxis, a widespread allergic reaction occurs throughout thevascular system and in closely associated tissues. Body-widevasodilation as well as increased permeability of the capillaries withresultant marked loss of plasma from the circulation occurs. Death mayresult from circulatory shock within minutes. In addition, leukotrienesare released which may elicit a massive asthma attack and death bysuffocation.

Urticaria results when an antigen enters specific skin areas and causeslocal anaphylactoid reactions and skin swelling commonly known as“hives.”

In hay fever, the allergic reaction occurs in the nose. Histaminereleased in response to the reaction causes local vascular dilation,with resultant increased capillary pressure as well as increasedcapillary permeability. Both of these effects cause rapid fluid leakageinto the tissues of the nose and the nasal linings become swollen andsecretory.

Asthma is characterized by spastic contraction of the smooth muscle inthe bronchioles, which causes extreme difficulty in breathing. Asthmaoccurs in 3 to 5 percent of all people at some time in their life. Theusual cause of asthma is hypersensitivity of the bronchioles to foreignsubstances in the air. About 70 percent of the asthma which occurs inyounger patients, i.e., those under 30 years of age, is caused byallergic hypersensitivity, in particular, by sensitivity to plantpollens. In older persons, the cause of asthma is almost alwayshypersensitivity to non-allergic type irritants such as air pollutionand the like.

The three hallmark features of allergic disease are the presence ofexcessive mast cells and eosinophils, and the production of IgE. Thecytokines responsible for this activity are interleukin 4 (IL-4) for IgEproduction (Finkelman et al., 1986, Proc. Natl. Acad. Sci. USA83:9675-9678; Coffman et al., 1986, J. Immunol. 136:949-954), IL-5 inthe case of eosinophilia (Sanderson et al., 1986, Proc. Natl. Acad. Sci.USA 83:437-440), and the combination of IL-3, IL-4, and IL-10 in thecase of mast cell production (Thompson-Snipes et al., 1991, J. Exp. Med.173:507-510).

The initiation of the immune response to a pathogen requires a complexseries of interactions among certain cell populations generallyinvolving cytokine production as reviewed by Gause and Lu (1996, In:Cytokine Regulation of Humoral Immunity, Snapper, ed., John Wiley andSons, New York). Within hours after immunization with a pathogen, ahighly pronounced and restricted cytokine pattern is detectable inlymphoid organs. Generally, the immune response may be classifiedaccording to the CD4+ T-helper (Th) cells associated therewith as eithera type 1 response, mediated by T-helper type 1 cells (Th1), or type 2response, mediated by T-helper type 2 cells. More recently, it has beendemonstrated that the type 1 and type 2 responses may each be mediatedby cells other than Th1 or Th2.

The type 1 response involves Th1-type cytokines including IFN-γ and IL-2and, since IFN-γ is a mediator for activation of macrophages andmonocytes, the type 1 response is associated with cellular immunity andinflammation. In contrast, Th2 cells are mediators of Ig production(humoral immunity) and produce IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13(Tanaka et al., 1996, In: Cytokine Regulation of Humoral Immunity, pp.251-272, Snapper, ed., John Wiley and Sons, New York). However, thecellular and humoral immunity compartments are intertwined asexemplified by the role of IL-6 in both responses which is mediated bycomplex interactions between various cells and the cytokines producedthereby. It has been demonstrated that interaction of Th2 cells with Bcells induces a humoral response (characterized by Ig production) inthat IL-4 signaling through CD40 induces IL-6 production which enhancesIg synthesis. In contrast, interaction of Th1 cells with macrophages ormonocytes causes production of, inter alia, IL-6 which, in turn, causesan inflammatory response. Therefore, certain cytokines may play a rolein both humoral and cellular immune responses and the production ofcytokines orchestrates a highly complex series of responses.

Of the humoral responses, the immune response mediated by IgE has beenthe most studied perhaps due to the fact that IgE mediates a unique andpotent set of effector functions that are central features of allergyand asthma. The principal cellular pathway leading to IgE productioninvolves B-cell activation via CD40-IL-4 signaling causing B cells toclass switch to IgE. In mice, there is compelling evidence that IL-4 isrequired for virtually all primary IgE responses. More specifically,treatment of mice with an excess of anti-IL-4 neutralizing antibodyinhibits 95-99% of primary IgE response to various stimuli (Finkelman etal., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:9675-9678; Finkelman et al.,1988, J. Immunol. 141:2335-2341; Finkelman et al., 1988, J. Immunul.140:1022-1027). Additionally, IFN-γ and antibodies to IL-5 and IL-4inhibit pulmonary eosinophilia in allergic mice sensitized by ovalbuminchallenge (Kung et al., 1995, Inflamm. Res. 44:S185-S186). Further,homozygous deletion transgenic mice lacking the IL-4 gene have onlytrace amounts of IgE after infection with various pathogens known tostimulate IgE production (Kuhn et al., 1991, Science 254:707-710; vonder Weid et al., 1994, Eur. J. Immunol. 24:2285-2293). Also, Lee et al.(1997, J. Exp. Med. 185:2143-2156) demonstrated that transgenic micewhich constitutively express IL-5 develop many of the pathologiesassociated with asthmatic patients including eosinophil invasion ofperibronchial spaces, epithelial hypertrophy, goblet cell hyperplasia,increased mucus production, and exhibit eosinophil recruitment to theairway lumen at levels comparable to asthmatic patients. Thus, thesestudies confirm the crucial role of IL-4 and other type 2 cytokines inIgE switching in mice and, more importantly, in the etiology of allergicdisease.

Although the role of IL-4 in IgE switching and/or the development ofallergic disease in humans is not as clear as it is in mice, the datasuggest that IL-4 and/or IL-13 are the major inducers of IgE switchingin humans; further, Th2-like type 2 responses have been demonstrated ina variety of allergic and parasitic diseases (Sher and Coffman, 1992,Annu. Rev. Immunol. 10:385-409; Yssel et al., 1992, J. Immunol.148:738-745; Romagnani, 1994, Annu. Rev. Immunol. 12:227-257; Wierengaet al., 1990, J. Immunol. 144:4651-4656; Coffman, 1996, In: CytokineRegulation of Humoral Immunity, pp. 379-389, Snapper, ed., John Wileyand Sons, New York). In addition, the prior art suggests that variousdisease conditions are associated with type 2 cytokine abnormalities assummarized by Hagiwara and Klinman (1996, In: Cytokine Regulation ofHumoral Immunity, pp. 409-430, Snapper, ed., John Wiley and Sons, NewYork). For instance, Kay et al. (1997, Int. Arch. Allergy Immunol.113:196-199), demonstrated that IL-4 and IL-5 production by eosinophilsmay amplify local allergic inflammatory responses in humans. Therefore,within the complex and interactive cytokine network which regulates themagnitude, nature and duration of immune responses against self andforeign antigens, there is mounting evidence of the role of the Th2-typecytokines, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13, in variousdisease states in humans including asthma.

The allergic reaction that occurs in the allergic type of asthma isbelieved to occur in the following manner. The typically allergic personhas a tendency to synthesize large amounts of IgE antibodies. In asthma,these antibodies are mainly attached to mast cells in the lunginterstitium in close association with the bronchioles and smallbronchi. When pollen is inhaled by a person hypersensitive to thepollen, (i.e., to which the person has already developed IgEantibodies), the pollen reacts with the mast cell-attached antibodiesand causes the cells to release several substances including histamine,leukotrines, eosinophilic chemotactic factor, and bradykinin. Thecombined results of the release of these factors is the production oflocalized edema in the walls of the bronchioles as well as secretion ofthick mucus into bronchiolar lumens, and spasm of the bronchiolar smoothmuscle causing the airway resistance to increase markedly.

The majority of treatments for allergies in humans involves theadministration of compounds which are directed to neutralizing theeffects of the substances released from mast cells or basophils. Thus, aplethora of antihistamines and other compounds are available whichneutralize the effects of histamines and the like following theirrelease from mast cells or basophils. The drawbacks of such treatmentsis the necessity that they are used essentially subsequent to theallergic event and do not prevent future allergic events in the patient.

Current treatment for asthma includes the administration of compoundsthat control the airway inflammatory component of the disease, e.g.,primarily corticosteroids, sodium cromolyn, methylxanthines andleukotriene pathway modifiers (see, e.g., Drazen et al., 1999, New Eng.J. Med. 340:197-206). In addition, there are available rapid reliefcompositions that counteract bronchospasm, e.g., primarilybeta-adrenergic agents. These compounds have several disadvantages inthat there is a risk that they will not be effective and despite theiradministration, the asthma attack will continue. In addition, severalside effects are associated with prolonged use of these type ofcompounds, particularly in the case of corticosteroids andbeta-adrenergic agents; further, there is a progressive loss ofsensitivity to these treatments after prolonged use. In severe asthma,these compounds are only of limited efficacy. Further, these compoundsare non-selective, i.e., they do not specifically target the lung,therefore, side-effects affecting other organs are a potential risk. Inaddition, there is an increasing body of evidence which indicates theremay be an increased risk of dying from bronchial asthma followingprolonged treatment of asthma using long-acting beta-adrenergic agentssuch as fenoterol (Pearce et al., 1990, Thorax 45:170-175; Spitzer etal., 1992, New Engl. J. Med. 326:560-561).

Approximately fifteen million individuals in the U.S. suffer fromasthma, and the disease is the cause of more than five thousand deathsannually in the U.S. In children, asthma represents the most prevalentchronic disease, requiring the most frequent use of emergency roomvisits and hospitalizations. The overall annual cost for asthma care inthe U.S. is estimated to be about nine billion dollars. Asthma is themost common cause of school and work absenteeism in the U.S.

To date, there are no long-term preventative treatments available forallergies in humans. Given the fact that allergic responses, andparticularly asthma, are on the rise in the human population, there is along felt need for the development of therapies which are designed toprevent as well as treat an allergic response in a human patient. Thepresent invention satisfies this need.

BRIEF SUMMARY OF THE INVENTION

The invention includes an allergy vaccine comprising at least oneprotein selected from the group consisting of an IgE, an IgE receptor,an interleukin involved in production of IgE in a mammal, a receptor foran interleukin involved in production of IgE in a mammal, aninterferon-alpha, an interferon-alpha receptor, a histamine, a histaminereceptor, a leukotriene, a leukotriene receptor, in apharmaceutically-acceptable carrier.

In one aspect, the mammal is selected from the group consisting of ahuman, a non-human primate, a horse, a cow, a pig, a goat, a dog, a cat,a rodent.

In a further aspect, the mammal is a human.

In another aspect, the interleukin is selected from the group consistingof IL-3, IL-4, IL-5, IL-6, IL-10, and IL-13.

In yet another aspect, the interleukin is selected from the groupconsisting of IL-3, IL-4, IL-5, IL-6, and IL-13.

In another aspect, the interleukin is selected from the group consistingof IL-3, IL-4, IL-5, and IL-13.

In yet another aspect, the interleukin is selected from the groupconsisting of IL-4, IL-5, and IL-13.

In a further aspect, the interleukin is selected from the groupconsisting of IL-4 and IL-5.

In yet a further aspect, the interleukin is IL-4.

In another aspect, the vaccine further comprises interferon gamma.

In a further aspect, the interleukin receptor is selected from the groupconsisting of an IL-3 receptor, an IL-4 receptor, an IL-5 receptor, anIL-6 receptor, an IL-10 receptor, and an IL-13 receptor.

The invention also includes an allergy vaccine comprising at least oneingredient selected from the group consisting of an interleukin involvedin production of IgE in a mammal, and a receptor for an interleukininvolved in production of IgE in a mammal, in apharmaceutically-acceptable carrier.

In one aspect, the vaccine further comprising interferon gamma.

The invention further includes an allergy vaccine comprising at leastone ingredient selected from the group consisting of an IgE, an IgEreceptor, an interferon-alpha, an interferon-alpha receptor, ahistamine, a histamine receptor, a leukotriene, and a leukotrienereceptor, in a pharmaceutically-acceptable carrier.

In one aspect, the vaccine further comprising interferon gamma.

The invention includes an allergy vaccine comprising at least oneisolated nucleic acid encoding a protein selected from the groupconsisting of an IgE, an IgE receptor, an interleukin involved inproduction of IgE in a mammal, a receptor for an interleukin involved inproduction of IgE in a mammal, an interferon-alpha, an interferon-alphareceptor, a histamine, a histamine receptor, a leukotriene, aleukotriene receptor, in a pharmaceutically-acceptable carrier.

In one aspect, the vaccine further comprises interferon gamma.

In another aspect, the isolated nucleic acid further comprises apromoter/regulatory sequence operably linked thereto.

In yet another aspect, the isolated nucleic acid further comprises avector.

The invention further includes a method of preventing an allergicresponse in a mammal. The method comprises administering to the mammalan allergy vaccine comprising at least one ingredient selected from thegroup consisting of an IgE, an IgE receptor, an interleukin involved inthe production of IgE in a mammal, a receptor for an interleukininvolved in the production of IgE in a mammal, an interferon alpha, aninterferon alpha receptor, an interferon-alpha, an interferon-alphareceptor, a histamine, a histamine receptor, a leukotriene, and aleukotriene receptor, thereby preventing an allergic response in amammal.

In one aspect, the interleukin is selected from the group consisting ofIL-3, IL-4, IL-5, IL-6, IL-10, and IL-13.

In another aspect, the interleukin is selected from the group consistingof IL-3, IL-4, IL-5, IL-6, and IL-13.

In yet another aspect, the interleukin is selected from the groupconsisting of IL-3, IL-4, IL-5, and IL-13.

In another aspect, the interleukin is selected from the group consistingof IL-4, IL-5, and IL-13.

In a further aspect, the interleukin is selected from the groupconsisting of IL-4 and IL-5.

In yet another aspect, the interleukin is IL-4.

In another aspect, the method further comprises administering interferongamma to the mammal.

The invention includes a method of preventing an allergic response in amammal. The method comprises administering to the mammal an allergyvaccine comprising at least one nucleic acid encoding an ingredientselected from the group consisting of an IgE, an IgE receptor, aninterleukin involved in the production of IgE in a mammal, a receptorfor an interleukin involved in the production of IgE in a mammal, aninterferon-alpha, an interferon-alpha receptor, a histamine, a histaminereceptor, a leukotriene, and a leukotriene receptor, thereby preventingan allergic response in a mammal.

In one aspect, the isolated nucleic acid further comprises apromoter/regulatory sequence operably linked thereto.

In another aspect, the isolated nucleic acid further comprises a vector.

In yet another aspect, the method further comprises administeringinterferon gamma to the mammal.

The invention also includes a method of treating an allergy in a mammal.The method comprises administering to the mammal an allergy vaccinecomprising at least one ingredient selected from the group consisting ofan interleukin involved in the production of IgE in a mammal, and areceptor for an interleukin involved in the production of IgE in amammal, thereby treating an allergy in a mammal.

In one aspect, the interleukin is selected from the group consisting ofIL-3, IL-4, IL-5, IL-6, IL-10, and IL-13.

In another aspect, the method further comprises administering interferongamma to the mammal.

The invention further includes a method of treating an allergy in amammal. The method comprises administering to the mammal an allergyvaccine comprising at least one ingredient selected from the groupconsisting of IgE, an IgE receptor, an interferon-alpha, aninterferon-alpha receptor, a histamine, a histamine receptor, aleukotriene, and a leukotriene receptor, thereby treating an allergy ina mammal.

In one aspect, the method further comprises administering interferongamma to the mammal.

The invention includes a method of preventing an allergic response in amammal. The method comprises administering to the mammal at least oneingredient selected from the group consisting of an anti-interleukincompound which is not an anti-interleukin antibody, wherein theinterleukin is at least one interleukin involved in the production ofIgE in a mammal, thereby preventing an allergic response in a mammal.

In one aspect, the anti-interleukin compound is selected from the groupconsisting of a soluble interleukin receptor, an d an antibody to aninterleukin receptor.

In another aspect, the interleukin involved in the production of IgE isselected from the group consisting of IL-3, IL-4, IL-5, IL-6, IL-10, andIL-13.

In yet another aspect, the method further comprises administeringinterferon gamma to the mammal.

The invention includes a method of treating an allergy in a mammal. Themethod comprises administering to the mammal at least one ingredientselected from the group consisting of an anti-IgE antibody, an anti-IgEreceptor antibody, a soluble IgE receptor, an anti-interleukin receptorantibody, a soluble interleukin receptor, an anti-interferon-alphaantibody, an anti-interferon-alpha receptor antibody, a solubleinterferon-alpha receptor, an anti-histamine antibody, an anti-histaminereceptor antibody, a soluble histamine receptor, an anti-leukotrieneantibody, an anti-leukotriene receptor antibody, and a solubleleukotriene receptor, wherein the interleukin is at least oneinterleukin involved in the production of IgE in a mammal, therebytreating an allergy in a mammal.

In one aspect, the interleukin involved in the production of IgE isselected from the group consisting of IL-3, IL-4, IL-5, IL-6, IL-10 andIL-13.

In a further aspect, the interleukin receptor is selected from the groupconsisting of an IL-3 receptor, an IL-4 receptor, an IL-5 receptor, anIL-6 receptor, and IL-10 receptor, and an IL-13 receptor.

In another aspect, the antibody to an interleukin receptor is selectedfrom the group consisting of an antibody to an IL-3 receptor, anantibody to an IL-4 receptor, an antibody to an IL-5 receptor, anantibody to an IL-6 receptor, an antibody to an IL-10 receptor, and anantibody to an IL-13 receptor.

In yet another aspect, the method further comprises administeringinterferon gamma to the mammal.

The invention includes a method of treating an allergy in a mammal. Themethod comprises administering to the mammal at least one antisensenucleic acid complementary to a nucleic acid encoding a protein selectedfrom the group consisting of IgE, an IgE receptor, an interleukininvolved in the production of IgE in a mammal, a receptor to aninterleukin involved in the production of IgE in a mammal, aninterferon-alpha, an interferon-alpha receptor, a histamine, a histaminereceptor, a leukotriene, and a leukotriene receptor, thereby treating anallergy in a mammal.

In one aspect, the interleukin is selected from the group consisting ofIL-3, IL-4, IL-5, IL-6, IL-10 and IL-13.

In another aspect, the method further comprises administering interferongamma to the mammal.

The invention includes a kit for preventing an allergic response in amammal. The kit comprises at least one allergy vaccine wherein theallergy vaccine comprises a protein selected from the group consistingof IgE, an IgE receptor, an interleukin involved in the production ofIgE, a receptor for an interleukin involved in the production of IgE, aninterferon-alpha, an interferon-alpha receptor, a histamine, a histaminereceptor, a leukotriene, and a leukotriene receptor, an applicator, andan instructional material for the use thereof.

In one aspect, the kit further comprises interferon gamma.

The invention also includes a kit for preventing an allergic response ina mammal. The kit comprises an allergy vaccine wherein the allergyvaccine comprises at least one nucleic acid encoding IgE, an IgEreceptor, an interleukin involved in the production of IgE, a receptorfor an interleukin involved in the production of IgE, aninterferon-alpha, an interferon-alpha receptor, a histamine, a histaminereceptor, a leukotriene, and a leukotriene receptor, wherein theinterleukin is at least one interleukin involved in the production ofIgE, an applicator, and an instructional material for the use thereof.

In one aspect, the kit further comprises interferon gamma.

The invention includes a kit for treating an allergy in a mammal. Thekit comprises an at least one ingredient selected from an anti-IgEantibody, an anti-IgE receptor antibody, a soluble IgE receptor, ananti-interleukin receptor antibody, a soluble interleukin receptor, ananti-interferon-alpha antibody, an anti-interferon-alpha receptorantibody, a soluble interferon-alpha receptor, an anti-histamineantibody, an anti-histamine receptor antibody, a soluble histaminereceptor, an anti-leukotriene antibody, an anti-leukotriene receptorantibody, and a soluble leukotriene receptor, wherein the interleukin isat least one interleukin involved in the production of IgE in a mammal,an applicator, and an instructional material for the use thereof.

In one aspect, the kit further comprises interferon gamma.

The invention includes a kit for treating an allergy in a mammal. Thekit comprises an antisense nucleic acid complementary to a nucleic acidencoding at least one ingredient selected from the group consisting ofIgE, an IgE receptor, an interleukin involved in the production of IgEin a mammal, a receptor to an interleukin involved in the production ofIgE in a mammal, an interferon-alpha, an interferon-alpha receptor, ahistamine, a histamine receptor, a leukotriene, and a leukotrienereceptor, an applicator, and an instructional material for the usethereof.

In one aspect, the kit further comprises interferon gamma.

The invention further includes a method of inhibiting production of IgEin a mammal. The method comprises administering to the mammal at leastone ingredient selected from the group consisting of an IgE, an IgEreceptor, an interleukin involved in the production of IgE in a mammal,a receptor for an interleukin involved in the production of IgE in amammal, an interferon-alpha, an interferon-alpha receptor, a histamine,a histamine receptor, a leukotriene, and a leukotriene receptor, therebyinhibiting IgE production in a mammal.

In one aspect, the interleukin is selected from the group consisting ofIL-3, IL-4, IL-5, IL-6, IL-10, and IL-13.

In another aspect, the method further comprises administering interferongamma to the mammal.

The invention includes a method of inhibiting production of IgE in amammal. The method comprises administering to the mammal at least onenucleic acid encoding a protein selected from the group consisting of anIgE, an IgE receptor, an interleukin involved in the production of IgEin a mammal, a receptor for an interleukin involved in the production ofIgE in a mammal, an interferon-alpha, an interferon-alpha receptor, ahistamine, a histamine receptor, a leukotriene, and a leukotrienereceptor, thereby inhibiting IgE production in a mammal.

In one aspect, the method further comprises administering interferongamma to the mammal.

The invention also includes a method of inhibiting production of IgE ina mammal. The method comprises administering to a mammal at least oneingredient selected from the group consisting of an anti-IgE antibody,an anti-IgE receptor antibody, a soluble IgE receptor, ananti-interleukin receptor antibody, a soluble interleukin receptor, ananti-interferon-alpha antibody, an anti-interferon-alpha receptorantibody, a soluble interferon-alpha receptor, an anti-histamineantibody, an anti-histamine receptor antibody, a soluble histaminereceptor, an anti-leukotriene antibody, an anti-leukotriene receptorantibody, and a soluble leukotriene receptor, wherein the interleukin isat least one interleukin involved in the production of IgE in a mammal,thereby inhibiting production of IgE in a mammal.

In one aspect, the interleukin is selected from the group consisting ofIL-3, IL-4, IL-5, IL-6, IL-10 and IL-13.

In another aspect, the method further comprises administering interferongamma to the mammal.

The invention further includes a method of inhibiting production of IgEin a mammal. The method comprises administering to the mammal at leastone antisense nucleic acid complementary to a nucleic acid encoding aprotein selected from the group consisting of IgE, an IgE receptor, aninterleukin involved in the production of IgE in a mammal, a receptor toan interleukin involved in the production of IgE in a mammal, aninterferon-alpha, an interferon-alpha receptor, a histamine, a histaminereceptor, a leukotriene, and a leukotriene receptor, thereby inhibitingproduction of IgE in a mammal.

In one aspect, the interleukin is selected from the group consisting ofIL-3, IL-4, IL-5, IL-6, IL-10 and IL-13.

In another aspect, the method further comprises administering interferongamma to the mammal.

The invention includes a kit for inhibiting production of IgE in amammal. The kit comprises at least one protein selected from the groupconsisting of IgE, an IgE receptor, an interleukin involved in theproduction of IgE, a receptor for an interleukin involved in theproduction of IgE, an interferon-alpha, an interferon-alpha receptor, ahistamine, a histamine receptor, a leukotriene, and a leukotrienereceptor, an applicator, and an instructional material for the usethereof.

In one aspect, the kit further comprises interferon gamma.

The invention also includes a kit for inhibiting production of IgE in amammal. The kit comprises an isolated nucleic acid encoding at least oneprotein selected from the group consisting of IgE, an IgE receptor, aninterleukin involved in the production of IgE, a receptor for aninterleukin involved in the production of IgE, an interferon-alpha, aninterferon-alpha receptor, a histamine, a histamine receptor, aleukotriene, and a leukotriene receptor, wherein the interleukin is atleast one interleukin involved in the production of IgE, an applicator,and an instructional material for the use thereof.

In one aspect, the kit further comprises interferon gamma.

The invention further includes a kit for inhibiting production of IgE ina mammal. The kit comprises at least one ingredient selected from ananti-IgE antibody, an anti-IgE receptor antibody, a soluble IgEreceptor, an anti-interleukin receptor antibody, a soluble interleukinreceptor, an anti-interferon-alpha antibody, an anti-interferon-alphareceptor antibody, a soluble interferon-alpha receptor, ananti-histamine antibody, an anti-histamine receptor antibody, a solublehistamine receptor, an anti-leukotriene antibody, an anti-leukotrienereceptor antibody, and a soluble leukotriene receptor, wherein theinterleukin is at least one interleukin involved in the production ofIgE in a mammal, an applicator, and an instructional material for theuse thereof.

In one aspect, the kit further comprises interferon gamma.

The invention includes a kit for inhibiting production of IgE in amammal. The kit comprises an antisense nucleic acid complementary to anucleic acid encoding at least one ingredient selected from the groupconsisting of IgE, an IgE receptor, an interleukin involved in theproduction of IgE in a mammal, a receptor to an interleukin involved inthe production of IgE in a mammal, an interferon-alpha, aninterferon-alpha receptor, a histamine, a histamine receptor, aleukotriene, and a leukotriene receptor, an applicator, and aninstructional material for the use thereof.

In one aspect, the kit further comprises interferon gamma.

The invention also includes a kit for preventing an allergic response ina mammal. The kit comprises a pharmaceutical composition comprising atleast one allergy vaccine in an amount effective for preventing anallergic response in a mammal in a pharmaceutically acceptable carrier,wherein the allergy vaccine comprises a protein selected from the groupconsisting of IgE, an IgE receptor, an interleukin involved in theproduction of IgE, a receptor for an interleukin involved in theproduction of IgE, an interferon-alpha, an interferon-alpha receptor, ahistamine, a histamine receptor, a leukotriene, and a leukotrienereceptor, an applicator, and an instructional material for the usethereof.

In one aspect, the kit further comprises interferon gamma.

The invention includes a kit for preventing an allergic response in amammal. The kit comprises a pharmaceutical composition comprising anallergy vaccine in a pharmaceutically acceptable carrier in an amounteffective for preventing an allergic response in a mammal, wherein theallergy vaccine comprises at least one nucleic acid encoding IgE, an IgEreceptor, an interleukin involved in the production of IgE, a receptorfor an interleukin involved in the production of IgE, aninterferon-alpha, an interferon-alpha receptor, a histamine, a histaminereceptor, a leukotriene, and a leukotriene receptor, wherein theinterleukin is at least one interleukin involved in the production ofIgE, an applicator, and an instructional material for the use thereof.

In one aspect, the kit further comprises interferon gamma.

The invention further includes a kit for treating an allergy in amammal. The kit comprising a pharmaceutical composition comprising atleast one protein in an amount effective for treating an allergy in amammal in a pharmaceutically acceptable carrier, wherein the protein isselected from an anti-IgE antibody, an anti-IgE receptor antibody, asoluble IgE receptor, an anti-interleukin receptor antibody, a solubleinterleukin receptor, an anti-interferon-alpha antibody, ananti-interferon-alpha receptor antibody, a soluble interferon-alphareceptor, an anti-histamine antibody, an anti-histamine receptorantibody, a soluble histamine receptor, an anti-leukotriene antibody, ananti-leukotriene receptor antibody, and a soluble leukotriene receptor,wherein the interleukin is at least one interleukin involved in theproduction of IgE in a mammal, an applicator, and an instructionalmaterial for the use thereof.

In one aspect, the kit further comprises interferon gamma.

The invention also includes a kit for treating an allergy in a mammal.The kit comprises a pharmaceutical composition comprising an antisensenucleic acid in an amount effective for treating an allergy in a mammalin a pharmaceutically acceptable carrier, wherein the antisense nucleicacid is complementary to a nucleic acid encoding at least one ingredientselected from the group consisting of IgE, an IgE receptor, aninterleukin involved in the production of IgE in a mammal, a receptor toan interleukin involved in the production of IgE in a mammal, aninterferon-alpha, an interferon-alpha receptor, a histamine, a histaminereceptor, a leukotriene, and a leukotriene receptor, an applicator, andan instructional material for the use thereof.

In one aspect, the kit further comprising interferon gamma. Theinvention includes a pharmaceutical composition comprising at least oneprotein selected from the group consisting of an IgE, an IgE receptor,an interleukin involved in production of IgE in a mammal, a receptor foran interleukin involved in production of IgE in a mammal, aninterferon-alpha, an interferon-alpha receptor, a histamine, a histaminereceptor, a leukotriene, a leukotriene receptor, in apharmaceutically-acceptable carrier in an amount effective forpreventing an allergic response in a mammal.

In one aspect, the composition further comprises interferon gamma.

The invention includes a pharmaceutical composition comprising at leastone isolated nucleic acid encoding a protein selected from the groupconsisting of an IgE, an IgE receptor, an interleukin involved inproduction of IgE in a mammal, a receptor for an interleukin involved inproduction of IgE in a mammal, an interferon-alpha, an interferon-alphareceptor, a histamine, a histamine receptor, a leukotriene, aleukotriene receptor, in a pharmaceutically-acceptable carrier, in anamount effective for preventing an allergic response in a mammal.

In one aspect, the composition further comprises interferon gamma.

The invention includes a pharmaceutical composition comprising at leastone protein in an amount effective for treating an allergy in a mammal,wherein the protein is selected from the group consisting of an anti-IgEantibody, an anti-IgE receptor antibody, a soluble IgE receptor, ananti-interleukin receptor antibody, a soluble interleukin receptor, ananti-interferon-alpha antibody, an anti-interferon-alpha receptorantibody, a soluble interferon-alpha receptor, an anti-histamineantibody, an anti-histamine receptor antibody, a soluble histaminereceptor, an anti-leukotriene antibody, an anti-leukotriene receptorantibody, and a soluble leukotriene receptor, wherein the interleukin isat least one interleukin involved in the production of IgE in a mammal,in a pharmaceutically acceptable carrier.

In one aspect, the composition further comprises interferon gamma.

The invention further includes a method of treating an allergy in amammal. The method comprises administering to the mammal at least onefirst ingredient selected from the group consisting of an anti-IL-3antibody, an anti-IL-4 antibody, an anti-IL-5 antibody, an anti-IL-6antibody, an anti-IL-10 antibody, and an anti-IL-13 antibody, and atleast one second ingredient selected from the group consisting of ananti-IgE antibody, an anti-IgE receptor antibody, a soluble IgEreceptor, an anti-interleukin receptor antibody, a soluble interleukinreceptor, an anti-interferon-alpha antibody, an anti-interferon-alphareceptor antibody, a soluble interferon-alpha receptor, ananti-histamine antibody, an anti-histamine receptor antibody, a solublehistamine receptor, an anti-leukotriene antibody, an anti-leukotrienereceptor antibody, and a soluble leukotriene receptor, wherein theinterleukin is at least one interleukin involved in the production ofIgE in a mammal, thereby treating an allergy in a mammal.

In one aspect, the interleukin involved in the production of IgE isselected from the group consisting of IL-3, IL-4, IL-5, IL-6, IL-10 andIL-13.

In another aspect, the interleukin receptor is selected from the groupconsisting of an IL-3 receptor, an IL-4 receptor, an IL-5 receptor, anIL-6 receptor, and IL-10 receptor, and an IL-13 receptor.

In yet another aspect, the antibody to an interleukin receptor isselected from the group consisting of an antibody to an IL-3 receptor,an antibody to an IL-4 receptor, an antibody to an IL-5 receptor, anantibody to an IL-6 receptor, an antibody to an IL-10 receptor, and anantibody to an IL-13 receptor.

In a further aspect, the method further comprises administeringinterferon gamma to the mammal.

The invention includes a method of treating bronchial asthma in a human.The method comprises administering to the human at least one ingredientselected from the group consisting of an anti-interferon-alpha antibody,an anti-interferon-alpha receptor antibody, and a solubleinterferon-alpha receptor, thereby treating bronchial asthma in a human.

In one aspect, the method further comprising administering to the humanat least one ingredient selected from the group consisting of ananti-IgE antibody, an anti-IgE receptor antibody, and a soluble IgEreceptor.

In one aspect, the method further comprises administering to the humanat least one ingredient selected from the group consisting of ananti-histamine antibody, and anti-histamine receptor antibody, and asoluble histamine receptor.

In one aspect, the method further comprises administering interferongamma to the human.

In another aspect, the method further comprises administering to thehuman an anti-interleukin antibody wherein the interleukin is selectedfrom the group consisting of IL-3, IL-4, IL-5, IL-6, IL-10 and IL-13.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that it is possible toprevent the production of IgE in a mammal by administering a compoundthat will inhibit ligand/receptor interactions necessary for theproduction of IgE. The inhibition of IgE production, in turn,alleviates, prevents, and/or treats an allergic response in a mammal.

One skilled in the art would appreciate, based upon the disclosureprovided herein, that there are various ways to inhibit production of aninterleukin involved in the production of IgE. The present inventionencompasses these methods.

In one aspect, the present invention discloses methods for inducing theimmune system of a mammal to actively inhibit the ligand/receptorinteractions associated with production of IgE, i.e., an activeimmunization method. In another aspect, the present invention provides amethod comprising administering a substance to a mammal such that theligand/receptor interactions that mediate production of IgE areinhibited, i.e., a passive immunization method. Both of theseapproaches, which can be applied individually or in concert, are morefully discussed below.

Active Allergy Vaccine Compositions

Protein-based

The invention includes eliciting an immune response to varioussubstances (i.e., IgE, interleukins, histamines, leukotrienes, and theirreceptors) thereby inhibiting ligand/receptor interactions necessary forIgE production.

One skilled in the art would also appreciate that a naturally occurringcompound found in the body (e.g., IgE, interleukins, histamines,leukotrienes, and their receptors) may not trigger an immune responseand/or may generate an immune response which is not sufficient toinhibit the desired target ligand/receptor interactions. Therefore, asmore fully discussed below, the present invention encompasses methods ofrendering a naturally occurring compound immunogenic using, for example,methods well known in the art.

In one aspect, the invention includes an allergy vaccine comprising animmunogenic IgE useful for inducing an immune response (e.g., humoral,cellular, or both), which response inhibits IgE ligand/receptorinteractions necessary for development of asthma, or allergic orautoimmune conditions. One skilled in the art would appreciate, basedupon the disclosure provided herein, that inducing an immune response toIgE blocks the necessary IgE/IgE receptor interactions that mediate anallergic condition, disease, or reaction.

The invention further includes an allergy vaccine comprising animmunogenic IgE receptor useful for eliciting an immune response to thereceptor. Similarly to generating an immune response to IgE itself,induction of an immune response to an IgE receptor blocks the IgE/IgEreceptor interactions required for the production of IgE and therefore,such an immune response is useful for treating and/or preventing anallergy mediated by the effects of IgE.

Also encompassed in the invention is an allergy vaccine comprising oneor more interleukins which are necessary components in the IgEproduction pathway in a mammal (e.g., IL-3, IL-4, IL-5, IL-6, IL-10,IL-13). Such an allergy vaccine, by inducing an immune response againstone or more of these interleukins, which immune response renders theinterleukin non-functional, diminishes production of IgE in the mammal.The invention therefore includes generating an immune response byadministering at least one immunogenic interleukin.

Therefore, one skilled in the art would appreciate, based upon thedisclosure provided herein, that an allergy vaccine encompasses thefollowing combinations of interleukins involved in the IgE productionpathway. That is, preferably, IL-4 can be administered in concert withIL-5. Similarly, IL-4, IL-5 and IL-13 can be administered in concert.Likewise, IL-4, IL-5, IL-13 and IL-3 can be administered in concert.Further, IL-4, IL-5, IL-13, IL-3 and IL-6 can be administered inconcert. Moreover, IL-4, IL-5, IL-13, IL-6 and IL-10 can be administeredin concert. The invention also encompasses IL-3, IL-4, IL-5, IL-6, IL-10and IL-13 being administered in concert. The skilled artisan wouldunderstand, based upon the disclosure provided herein, that theinvention is not limited to these particular combination but encompassesan allergy vaccine comprising any combination of these interleukins.Further, the invention encompasses an allergy vaccine comprising theseinterleukins individually (e.g., IL-3 alone, IL-4 alone, IL-5 alone,IL-6 alone, IL-10 alone, and IL-13 alone).

Further, the invention encompasses an allergy vaccine comprising atleast one interleukin receptor. Inhibiting the interaction of aninterleukin involved in the IgE production pathway (e.g., IL-3, IL-4,IL-5, IL-6, IL-10, IL-13) by immunizing a mammal with an immunogenicinterleukin receptor is useful in that, as disclosed herein, antibodiesto an interleukin receptor are useful to inhibit interaction of theinterleukin receptor with its cognate interleukin ligand thus preventingnecessary receptor/ligand interactions in the IgE mediated immunepathway thereby inhibiting or decreasing IgE production.

In particular, it has been discovered that various combinations ofinterleukin receptors may be administered to a patient afflicted with anallergic disease or condition. Such combinations include: IL-4 receptorand IL-5 receptor; IL-4 receptor, IL-5 receptor and IL-13 receptor;IL-4-receptor, IL-5 receptor, IL-13 receptor and IL-3; IL-4 receptor,IL-5 receptor, IL-13 receptor, IL-3 receptor and IL-6 receptor; IL-4receptor, IL-5 receptor, IL-13 receptor, IL-6 receptor and IL-10receptor; and IL-3 receptor, IL-4 receptor, IL-5 receptor, IL-13receptor, IL-6 receptor and IL-10 receptor, to generate an immuneresponse to that receptor.

The invention encompasses administration of each receptor alone. Morespecifically, the invention includes administering IL-3 receptor alone,IL-4 receptor alone, IL-5 receptor alone, IL-6 receptor alone, IL-10receptor alone, and IL-13 receptor alone.

The invention includes an allergy vaccine comprising aninterferon-alpha, an interferon-alpha receptor, or both. This is becauseinterferon-alpha can exacerbate bronchial asthma attacks (see, e.g.,Bini et al., 1999, Mayo Clin. Proc. 74:367-370). Further, administrationof interferon-alpha to patients with mild asthma in order to treat otherdiseases or conditions in the patients resulted in exacerbation of theunderlying asthma (Krasnowska et al., 1992, Arch. Immunol. Ther. Exp.(Warsz) 40:75-78). In these patients, the severe asthmatic symptomsresolved upon discontinuation of interferon-alpha, but repeatedtreatment with interferon-alpha of these same patients several monthslater resulted in a rapid, more severe exacerbation of the asthma (id.).

These studies suggest that interferon-alpha mediates an allergic, e.g.,asthmatic, response in humans such that removal of interferon-alphaand/or inhibiting the interaction of interferon-alpha with its cognatereceptor inhibits or decreases an allergic response in a human.Therefore, the present invention encompasses an allergy vaccine thatgenerates an immune response to interferon-alpha, to its receptor, orboth, thereby inhibiting the necessary interferon-alpha ligand/receptorinteraction required to mediate an allergic response.

The invention further encompasses an allergy vaccine comprising ahistamine. One skilled in the art would appreciate, based upon thedisclosure provided herein, that inducing an immune response to ahistamine associated with the production of IgE prevents interactionsbetween histamine and histamine receptor thereby preventing thedevelopment of allergic response in a mammal otherwise mediated by suchinteractions.

Likewise, the present invention includes an allergy vaccine comprising ahistamine receptor, since, as discussed previously herein, an immuneresponse to such a receptor inhibits interactions between histamine andits receptor which would otherwise mediate IgE production. Preventingsuch interactions inhibits the development of an allergic responsemediated by IgE production.

In addition, the present invention encompasses an allergy vaccinecomprising at least one leukotriene. Such a vaccine prevents thedevelopment of allergic response in a mammal by inhibitingleukotriene/leukotriene receptor interactions necessary for such anallergic response.

Similarly, the present invention includes an allergy vaccine comprisingat least one leukotriene receptor since, as stated previously elsewhereherein, induction of an immune response to a leukotriene receptorinhibits the interactions between the leukotriene and its cognatereceptor such that the production of IgE dependent on such interactionsis also inhibited.

One skilled in the art would appreciate, based upon the disclosureprovided herein, that the invention encompasses an allergy vaccinecomprising at least one substance described previously (i.e., IgE,interleukin, interferon-alpha, histamine, leukotriene, and receptorsthereof) and any combination thereof Thus, the invention encompasses awide plethora of combinations as being an allergy vaccine including, butnot limited to, an allergy vaccine comprising IgE, IL-4, and IL-10, oran allergy vaccine comprising IL-5 receptor, histamine, a leukotrienereceptor, and interferon-alpha. Other combinations as would beunderstood by one skilled in the art based upon the disclosure providedherein are also included in the present invention.

Further, the aforementioned allergy vaccines further comprise apharmaceutically-acceptable carrier.

In addition, the present invention includes an allergy vaccine furthercomprising interferon gamma. Interferon gamma is useful for thetreatment of allergic disease in humans and inhibits many of the effectsof IL-4 on both murine and human B cells (see, e.g., King et al., 1989,Proc. Natl. Acad. Sci. USA 86:10085-10089; Snapper, 1996, In: CytokineRegulation of Humoral Immunity, pp. 324-346, Snapper, ed., John Wileyand Sons, New York; de Vries and Punnonen, Ibid. at pp. 195-215).Therefore, one skilled in the art would appreciate, based upon thedisclosure provided herein, that administration of interferon gamma incombination with the administration of at least one of theafore-mentioned substances (i.e., IgE, interleukin, histamine,leukotrienes, and/or their receptors), or any combination thereof, willfurther treat and/or alleviate an allergic disease or condition byfurther inhibiting the effect(s) of IL-4 involved in the disease processin the mammal thereby further reducing the level of IgE thus treatingand/or alleviating the allergy disease or condition.

DNA-based Allergy Vaccine

One skilled in the art would also appreciate, based upon the disclosureprovided herein, that an allergy vaccine (i.e., an immunogenic IgE, IgEreceptor, an interleukin involved in IgE production, an interleukinreceptor, an interferon-alpha, an interferon-alpha receptor, ahistamine, a histamine receptor, a leukotriene, a leukotriene receptor,or any combination thereof) can be administered either as a protein oras a nucleic acid encoding the protein. That is, it is well-known in theart that a nucleic acid encoding an immunogen may be administered as anaked DNA vaccine or may comprise a vector to generate an immuneresponse to the protein encoded by the nucleic acid (Seder andGurunathan, 1999, New. Eng. J. Med. 341:277-278).

The use of an isolated nucleic acid to generate an immune response,where the nucleic acid is in a vector (e.g., a plasmid or a virus), orwhere the nucleic acid comprises naked nucleic acid not associated withany other nucleic acid, is well-known in the art. For example, methodsfor construction of nucleic acid vaccines are described in Burger et al.(1991, J. Gen. Virol. 72:359-367), and are well-known in the art. Seealso Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, New York; Ausubel et al., 1997, CurrentProtocols in Molecular Biology, Green & Wiley, New York.

Briefly, an isolated nucleic acid encoding, for example, an IgE, an IgEreceptor, an interleukin involved in IgE production, and the like, maybe administered to a human in a pharmaceutically-acceptable carrier byintramuscular injection or via other routes as described elsewhereherein with respect to the administration of a polypeptide-based allergyvaccine. Additionally, the isolated nucleic acid may comprise a non-livevector such as, but not limited to, plasmid-based recombinants,heat-killed vaccines, liposomes, polyamine derivatives of DNA, and thelike, and also viral-based vectors such as for example, but withoutlimitation, adenovirus, poxvirus, herpesvirus, and adenovirus associatedvectors.

To express the isolated nucleic acid encoding an allergy vaccineprotein, the isolated protein-encoding nucleic acid sequence is operablylinked to a promoter/regulatory region capable of driving high levels ofexpression of the protein in cells. Many such promoter/regulatorysequences are available in the art including, but not limited to, forexample, the human cytomegalovirus immediate early promoter/enhancersequence, the SV40 early promoter, the Rous sarcoma virus promoter andother mammalian promoter/enhancer sequences. Moreover, inducible andtissue specific expression of the isolated nucleic acid operably linkedthereto may be accomplished by placing the nucleic acid under thecontrol of an inducible or tissue specific promoter/regulatory sequence.Examples of tissue specific or inducible promoter/regulatory sequenceswhich are useful for this purpose include, but are not limited to theMMTV long terminal repeat (LTR) inducible promoter, and the SV40 lateenhancer/promoter. In addition, promoters which are well known in theart which are induced in response to inducing agents such as metals,glucocorticoids, and the like, are also contemplated in the invention.Thus, it will be appreciated that the invention should be construed toinclude the use of any promoter/regulatory sequence which is eitherknown or is heretofore unknown, which is capable of driving expressionof the nucleic acid operably linked thereto.

Thus, the present invention encompasses an allergy vaccine comprising anucleic acid encoding at least one of an IgE, an IgE receptor, aninterleukin involved in IgE production, an interleukin receptor, aninterferon-alpha, an interferon-alpha receptor, a histamine, a histaminereceptor, a leukotriene, a leukotriene receptor, or any combinationthereof.

Active Immunization Methods

The present invention includes methods of treating an allergy and/orpreventing an allergic response in a mammal. The invention encompassesactive immunization methods encompassing administering to the mammal anallergy vaccine, i.e., a protein or peptide or the nucleic acid encodingthe protein or peptide that elicits an immune response which, in turn,inhibits necessary ligand/receptor interactions required for IgEproduction. Such allergy vaccines encompass IgE, IgE receptor, aninterleukin involved in IgE production (e.g., IL-3, IL-4, IL-5, IL-6,IL-10, IL-13), a receptor for such an interleukin, an interferon-alpha,a histamine, a leukotriene, their respective receptors. Additionally,the present invention encompasses that an allergy vaccine can beadministered individually or in combination, either alone or in concertwith interferon gamma.

Further, the present invention includes methods of treating an allergyand/or preventing an allergic response comprising administering anucleic acid encoding IgE, an interleukin involved in IgE production(e.g., IL-3, IL-4, IL-5, IL-6, IL-10, IL-13), an interferon-alpha, ahistamine, a leukotriene, and their receptors. The afore-mentionedproteins and/or nucleic acids encoding them can be administeredseparately or combined, and can be administered separately or in concertwith interferon gamma.

The main advantage of the allergy treatment of the invention over priorart treatments is that the present treatment offers long term relief,compared with the relatively short term relief provided by prior arttreatments. Further, unlike prior art treatments which are notpreventative and which only treat the allergic reaction once it hasoccurred, the present invention provides a method of prophylaxis thatprevents the unwanted immune response. In addition, the presentinvention provides treatments which circumvent the need for repeatedpatient compliance. That is, immunization methods do not require ongoingtreatment regimens which may make it difficult for patients to properlycomply. The lack of need for follow-up and/or ongoing office visits alsolimits the cost of the treatment as opposed to prior art methods.Moreover, as mentioned previously elsewhere herein, the instantinvention circumvents the drawbacks of prior art methods in thatunwanted side effects due to the non-specificity of the treatments arereduced or eliminated.

The types of allergy which can be treated using the compositions andmethods of the invention include, but are not limited to, asthma,anaphylaxis, hay fever and urticaria. Preferably, the allergy to betreated using the compositions and methods of the invention is asthma.

The present invention further encompasses methods of inhibiting theeffect(s) of an interleukin otherwise involved in production of IgE byinhibiting the effect(s) of a substance released by a cell activated byIgE (e.g., histamine and leukotrienes) by active immunization againstthe substance or its receptor(s) or by passive immunization byadministration of an antibody to the substance, an antibody to thereceptor(s) of the substance, and/or a soluble receptor of the substancewhich is not associated with a cell.

One skilled in the art would also appreciate, based upon the disclosureprovided herein, that the production of IgE may be further inhibited byadministering a cytokine which directly inhibits IgE production inaddition to the methods disclosed previously herein. Therefore, thepresent invention should be construed to include the administration of acytokine, preferably, IFNγ, IL-12, and the like, which directly inhibitsIgE production, in addition to the generation of an immune response toan interleukin involved in IgE production, the administration of ananti-interleukin compound, and/or the administration of an antisensenucleic acid all of which inhibit the production of an interleukin whichwould otherwise induce IgE production. The IFNγ is administered to amammal before, at the same time, or after the administration of theimmunogenic protein or nucleic acid encoding the same thereby furtherreducing the production of IgE in the human.

One skilled in the art would appreciate, based upon the disclosureprovided herein, that large quantities of these proteins can be producedusing a wide variety of methods well-known in the art. For instance,since the amino acid sequences of ILs and their receptors as well as thesequences of the nucleic acids encoding these molecules are known in theart (see, e.g., Snapper, 1996, In: Cytokine Regulation of HumoralImmunity, in passim, Snapper, ed., John Wiley and Sons, New York), largescale purification of ILs and IL-receptor proteins and/or nucleic acidsencoding them may be performed according to standard recombinant DNAtechniques such as those described, for example, in Sambrook et al.(1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor,N.Y.), and in Ausubel et al. (1997, Current Protocols in MolecularBiology, Green & Wiley, New York).

With respect to active immunization, individual or combinations of theproteins (e.g., IgE, interleukins, interferon-alpha, histamines,leukotrienes, and their receptors) can be obtained as describedpreviously elsewhere herein and may be rendered immunogenic using anynumber of techniques known in the art and described, for example, inHarlow et al. (1999, Using Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press, NY). That is, the allergy vaccine polypeptidesdisclosed herein for the treatment of allergy are naturally occurringcompounds found in the human body. For this reason, simply introducingany one of these compounds into a human may not trigger a humoral orcellular immune reaction. In order to elicit an immune reaction in ahuman to an IgE, an interleukin, an interferon-alpha, a histamine, aleukotriene, their receptors, and any combination thereof, whereadministered thereto, it is first necessary that the protein be renderedimmunogenic. This may be accomplished in several ways well known in theart of immunogenicity. For example, the protein may be treated with achemical which induces a modification of the protein which renders theprotein immunogenic such as treatment using diazabenzole (Obermyer andPick, 1903, Wien. Klin.Wschr. 16:659; 1904, Wien. Klin.Wschr.17:265).

Alternatively, the allergy vaccine protein of interest may be conjugatedto another composition, for example, a peptide, a hapten, or gelatin,which renders the interleukin immunogenic. While examples of methods ofrendering a protein of interest immunogenic are described herein, theinvention should not be construed to be limited to the specific methodsdisclosed, but rather, should be construed to include any and all known,or heretofore unknown, methods of rendering an interleukin, histamine,leukotriene, IgE, and their receptors, immunogenic.

The protein of interest (e.g., IgE, interleukin, interferon-alpha,histamine, leukotriene, and a receptor thereof) may be renderedimmunogenic (and may also be inactivated) by treatment with formalinessentially as described in Carelli et al. (1992, Biomed. andPharmacother. 46:149-153). Inactivation of a protein normally present ina human for use as an allergy vaccine to be administered to a human,while retaining the immunogenicity of the naturally-occurring protein,has the advantage of preventing the normal action of the introducedprotein preventing any side effects which may arise as a result of anexcess of the protein in the body. Nonetheless, an immune response,essential for the treatment of the desired allergy, is elicited.

In one aspect, the protein can be treated with formalin using thefollowing protocol. An amount of protein (e.g., about 1 mg) is admixedwith about 50 mg of human serum albumin and is dissolved in about 50 mlof sterile 70 mM Na₂HPO₄ solution, pH 8.22. The mixture is incubated forsix days at about 37° C. after which time it is added to about 8 pH ofsterile lysine, HCl solution (125 mg/ml). The solution is subsequentlydialyzed against about 20 times its volume of sterile PBS solution whichis diluted 1:10 (v/v).

The protein may also be rendered immunogenic by conjugation of theinterleukin to another composition, for example, bacterial tetanustoxoid protein essentially as described in Chu et al. (1983, Infn.Immun. 40:245-256) and in U.S. Pat. No. 4,902,506.

As is shown in the work of Obermeyer and Pick (1903, Wien. Klin.Wschr.16:659; 1904, Wien. Klin.Wschr.17:265), immunization of a mammal with aprotein treated with diazabenzole can induce an antibody which hasspecificity to diazabenzole and specificity to the protein connected tothe diazabenzole. Thus, treatment of interleukins and other proteins ofinterest with diazabenzole can induce antibodies which will react withthese interleukins and proteins in an autosystem and an isosystem.

It is not necessary that a full length protein be used as an immunogenin an allergy vaccine for the elicitation of an immune response.Fragments of protein that elicit an immune response which renders thebiologically active form of the subject protein inactive are alsocontemplated by the invention. Fragments of protein may be obtained bydegradation of full length molecule, by chemical synthesis, or bycloning and expression of such fragments using molecular biologytechnology described, for example, in Sambrook et al. (1989, MolecularCloning. A Laboratory Manual, Cold Spring Harbor, N.Y.), and in Ausubelet al. (1997, Current Protocols in Molecular Biology, Green & Wiley, NewYork). Once the DNA encoding the interleukin is known, it is a simplematter to obtain fragments of the interleukin by subcloning andexpressing DNA encoding a fragment of the interleukin.

Modifications of the protein (e.g., IgE, interleukin, interferon-alpha,histamine, leukotriene, and their receptors), or a fragment thereof, maybe made by direct modification of the protein or fragment thereof. Itwill be appreciated, of course, that a peptide may incorporate aminoacid residues which are modified without affecting activity (e.g., theimmunogenicity of the protein). For example, the terminal portion of thepeptide may be derivatized to include blocking groups, i.e., chemicalsubstituents suitable to protect and/or stabilize the N- and C-terminifrom “undesirable degradation”, a term meant to encompass any type ofenzymatic, chemical or biochemical breakdown of the compound at itstermini which is likely to affect the function of the compound as animmunogenic agent, i.e., sequential degradation of the interleukin at aterminal end thereof.

Blocking groups include protecting groups conventionally used in the artof peptide chemistry which will not adversely affect the in vivoactivities of the peptide. For example, suitable N-terminal blockinggroups can be introduced by alkylation or acylation of the N-terminus.Examples of suitable N-terminal blocking groups include C₁-C₅ branchedor unbranched alkyl groups, acyl groups such as formyl and acetylgroups, as well as substituted forms thereof, such as theacetamidomethyl (Acm) group. Desamino analogs of amino acids are alsouseful N-terminal blocking groups, and can either be coupled to theN-terminus of the peptide or used in place of the N-terminal reside.Suitable C-terminal blocking groups, in which the carboxyl group of theC-terminus is either incorporated or not, include esters, ketones oramides. Ester or ketone-forming alkyl groups, particularly lower alkylgroups such as methyl, ethyl and propyl, and amide-forming amino groupssuch as primary amines (—NH₂), and mono- and di-alkylamino groups suchas methylamino, ethylamino, dimethylamino, diethylamino,methylethylamino and the like are examples of C-terminal blockinggroups. Descarboxylated amino acid analogues such as agmatine are alsouseful C-terminal blocking groups and can be either coupled to thepeptide's C-terminal residue or used in place of it. Further, it will beappreciated that the free amino and carboxyl groups at the termini canbe removed altogether from the peptide to yield desamino anddescarboxylated forms thereof without affect on peptide activity.

Other modifications can also be incorporated without adversely affectingimmunogenic activity and these include, but are not limited to,substitution of one or more of the amino acids in the natural L-isomericform with amino acids in the D-isomeric form. Thus, the peptide mayinclude one or more D-amino acid resides, or may comprise amino acidswhich are all in the D-form. Retro-inverso forms of peptides inaccordance with the present invention are also contemplated, forexample, inverted peptides in which all amino acids are substituted withD-amino acid forms.

Acid addition salts of the present invention are also contemplated asfunctional equivalents. Thus, a peptide in accordance with the presentinvention treated with an inorganic acid such as hydrochloric,hydrobromic, sulfuric, nitric, phosphoric, and the like; or an organicacid such as an acetic, propionic, glycolic, pyruvic, oxalic, malic,malonic, succinic, maleic, fumaric, tartaric, citric, benzoic, cinnamic,mandelic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, salicylicand the like, to provide a water soluble salt of the peptide is suitablefor use as an immunogenic agent.

The present invention also provides for analogs of proteins or peptidesof interleukins. Analogs can differ from naturally occurring proteins orpeptides by conservative amino acid sequence differences or bymodifications which do not affect sequence, or by both.

For example, conservative amino acid changes may be made, which althoughthey alter the primary sequence of the protein or peptide, do notnormally alter its function. Conservative amino acid substitutionstypically include substitutions within the following groups:

-   -   glycine, alanine;    -   valine, isoleucine, leucine;    -   aspartic acid, glutamic acid;    -   asparagine, glutamine;    -   serine, threonine;    -   lysine, arginine;    -   phenylalanine, tyrosine.        Modifications (which do not normally alter primary sequence)        include in vivo, or in vitro chemical derivatization of        peptides, e.g., acetylation, or carboxylation. Also included are        modifications of glycosylation, e.g., those made by modifying        the glycosylation patterns of a peptide during its synthesis and        processing or in further processing steps; e.g., by exposing the        peptide to enzymes which affect glycosylation, e.g., mammalian        glycosylating or deglycosylating enzymes. Also embraced are        sequences which have phosphorylated amino acid residues, e.g.,        phosphotyrosine, phosphoserine, or phosphothreonine.

As described herein, also included are proteins and peptides which havebeen modified using ordinary molecular biological techniques so as toimprove their resistance to proteolytic degradation or to optimizesolubility properties or to render them more suitable as an immunogenicagent. Analogs of such peptides include those containing residues otherthan naturally occurring L-amino acids, e.g., D-amino acids ornon-naturally occurring synthetic amino acids. The peptides of theinvention are not limited to products of any of the specific exemplaryprocesses listed herein.

Further, one skilled in the art would appreciate, based upon thedisclosure provided herein, that the invention encompasses proteins andfragments and portions thereof where the portion or fragment elicits animmune response which blocks the activity of the naturally occurring,i.e., native, protein. Therefore, the invention encompasses full-lengthproteins, peptides, and also fragments and/or portions thereof, thatinduce an immune response that in turn, inhibits IgE production.

An allergy vaccine can comprise a sterile solution of modifiedinterleukins, histamine, leukotrienes and IgE, and/or their receptors,dissolved in 0.9% sodium chloride or sterile water. Final concentrationof interleukins, leukotrienes, histamine, IgE, and/or their receptors,is 100-200 micrograms/ml. Preferably, allergy vaccines are administeredintramuscularly or subcutaneously at dosages between 200 and 500micrograms once a week for 3 to 4 weeks, then once a month for 3additional months. Other schedules are possible such that more or lessfrequent immunizations can be administered over longer periods of time.

Typically, first immunization is given without an adjuvant, andsubsequent doses are administered with adjuvants. Based on clinicalresponse and immunologic parameters (e.g., antibody titers, avidity ofantibodies, antibody isotypes, and the like), further boosterimmunizations can be administered as deemed necessary.

Briefly, active immunization using interleukins can be performed asfollows. The initial titer of IgE is assessed. A high level of IgE is amarker of risk for allergy. Thus, a mammal exhibiting high titers of IgEwill be treated to prevent allergy.

The immunogen of choice (i.e., IgE, IgE-receptor, IL-3, IL-5, IL-6,IL-10, IL-13, receptor to IL-3, IL-5, IL-6, IL-10, IL-13,interferon-alpha, interferon -alpha receptor, histamine, histaminereceptor, leukotriene, leukotriene receptor) can be administeredapproximately 2-3 times/month.

In one aspect, IL-4 is administered alone or in combination with otherinterleukins (e.g., IL-3, IL-5, IL-6, IL-10, and IL-13). Eachinterleukin is administered at a dosage ranging from about 100 to 1000micrograms per dose. Preferably, the first dose is administered withoutadjuvant, but later doses are administered with adjuvant.

After the third injection, the level of, for instance, interleukins andIgE is assessed. Upon assessment, if improvement (e.g., IgE leveldecreases and/or the level of interleukin IL-4 and IL-5, and IL-13, orat least IL-4 and IL-5, decreases) is detected along with clinicalimprovement, then interleukin vaccinations continue to be administeredapproximately once per month for 3 months at a dose of approximately 500micrograms per injection.

If no improvement is detected according to standard laboratory assayand/or well-known clinical indices, interleukins are administered (IL-3,IL-6, IL-10) at a dose about 500 micrograms per injection.

If still no improvement is detected, i.e., in difficult cases,vaccination is performed using, interleukins and interferon gamma (nottreated by formalin or made immunogenic by other methods), at a dosagebetween about 1×10⁴ to 1×10⁸ units per square meter of body surfacearea.

The immune response to the immunogenic interleukin is measured bystandard immunological techniques such as ELISA or Western blotting andother such techniques well-known in the art or to be developed in thefuture. A variety of immunoassay formats may be used to selectantibodies specifically immunoreactive with a particular protein. See,e.g., Harlow et al. (1999, Using Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press, NY) for a description of immunoassayformats and conditions that can be used to determine specificimmunoreactivity.

In another aspect, IL-4 is administered in combination with anotherinterleukin involved in IgE production, and/or in combination with oneor more proteins selected from the group consisting of IgE, IgEreceptor, interleukin receptor, interferon-alpha, interferon-alphareceptor, histamine, histamine receptor, leukotriene, and leukotrienereceptor, in any combination.

In addition, the instant invention includes methods of treating orpreventing asthma, allergy, and autoimmune conditions comprisingadministering interferon gamma.

Passive Immunization Methods

In addition to the active immunization methods disclosed previouslyelsewhere herein, the present invention includes methods of treating anallergy and/or preventing an allergic response in a mammal comprisingpassively administering a protein (i.e., an anti-IgE antibody, ananti-IgE receptor antibody, a soluble IgE receptor, an anti-interleukinreceptor antibody, a soluble interleukin receptor, ananti-interferon-alpha antibody, an anti-interferon-alpha receptorantibody, a soluble interferon-alpha receptor, an anti-histamineantibody, an anti-histamine receptor antibody, a soluble histaminereceptor, an anti-leukotriene antibody, an anti-leukotriene receptorantibody, and a soluble leukotriene receptor) that directly inhibits IgEproduction without generating an immune response thereto.

The present invention does not encompass administering an anti-IL-4where the antibody is administered alone or with another antibody thatspecifically binds with IL-4 even if the antibodies recognize differentepitopes present in the IL-4 molecule.

Similarly, the present invention does not encompass administering ananti-IL-5 where the antibody is administered alone or with anotherantibody that specifically binds with IL-5 even if the antibodiesrecognize different epitopes present in the IL-5 molecule.

Nevertheless, the present invention includes administering anti-IL-4,anti-IL-5 antibody, alone or combined, in addition to administering atleast one protein selected from the group consisting of an anti-IgEantibody, an anti-IgE receptor antibody, a soluble IgE receptor, ananti-interleukin receptor antibody, a soluble interleukin receptor, ananti-interferon-alpha antibody, an anti-interferon-alpha receptorantibody, a soluble interferon-alpha, an anti-histamine antibody, ananti-histamine receptor antibody, a soluble histamine receptor, ananti-leukotriene antibody, an anti-leukotriene receptor antibody, and asoluble leukotriene receptor. Thus, the invention includes administeringanti-IL-4 and anti-IL-5, either separately or combined, in combinationwith one or more of the afore-mentioned proteins, or a fragment thereof.

The present invention includes passive immunization comprisingadministering an anti-interleukin antibody where the interleukin isinvolved in IgE production (e.g., IL-3, IL-4, IL-5, IL-6, IL-10, IL-13)together with another ingredient that inhibits a receptor-ligandinteraction involved in IgE production (e.g., an anti-IgE antibody, ananti-IgE receptor antibody, a soluble IgE receptor, an anti-interleukinreceptor antibody, a soluble interleukin receptor, ananti-interferon-alpha antibody, an anti-interferon alpha receptorantibody, a soluble interferon-alpha receptor, an anti-histamineantibody, an anti-histamine receptor antibody, a soluble histaminereceptor, an anti-leukotriene antibody, an anti-leukotriene receptorantibody, and a soluble leukotriene receptor).

The present invention also encompasses a passive immunization methodcomprising administering an antisense nucleic acid complementary to anucleic acid encoding IgE, an interleukin involved in IgE production(e.g., IL-3, IL-4, IL-5, IL-6, IL-10, IL-13), an interferon-alpha, ahistamine, a leukotriene, and/or an antisense nucleic acid complementaryto a nucleic acid encoding their respective receptors.

The invention encompasses administering any or all of these substanceseither alone or combined.

Further, the invention encompasses administering interferon gamma inconcert with at least one of the above-mentioned substances for passiveimmunization purposes.

Proteins

In addition to the active immunization methods disclosed previouslyelsewhere herein, the invention includes passive immunization methods ofinhibiting the action of IgE by administering to a mammal antibodies toIgE, antibodies to IgE receptor, and soluble IgE receptor. Further, inaddition to such passive immunization, the invention includesadministering to a mammal antibodies to an interleukin receptor wherethe interleukin mediates production of IgE (e.g., IL-3, IL-4, IL-5,IL-6, IL-10, IL-13), or soluble interleukin receptor thereby preventing,treating, or both, the development of allergic response in a mammal.

The invention further includes methods of inhibiting the action ofinterferon-alpha by administering to a mammal antibodies tointerferon-alpha, antibodies to interferon-alpha receptor, and solubleinterferon-alpha receptor.

Additionally, the invention includes a method of inhibiting the actionof histamine by administering to a mammal antibodies to histamine,antibodies to histamine receptor, and soluble histamine receptor.

Furthermore, the invention includes a method of inhibiting the action ofleukotrienes by administering to a mammal antibodies to leukotrines,antibodies to leukotriene receptor, and soluble leukotriene receptor.

One skilled in the art would appreciate, based upon the disclosureprovided herein, that the invention encompasses proteins, peptides, andfragments or portions thereof, which when administered, inhibitproduction of IgE. That is, the invention is not limited to passiveimmunization using the full-length protein. The invention alsoencompasses any portion or fragment of a protein involved in IgEproduction (e.g., IgE, interleukins IL-3, IL-4, IL-5, IL-6, IL-10, andIL-13, interferon-alpha, histamine, leukotriene, and their receptors,and/or antibodies thereto) that is capable of blocking a ligand/receptorinteraction involved in IgE production

The decision to use active or passive immunization is one which will bemade by the skilled artisan and will depend upon any number of factorsincluding, but not limited to, the type of allergy being treated, theseverity of the disease and the age and overall health of the individualbeing treated.

Further, the invention includes passive immunization of patients withplasma or IgG from donors immunized with receptors to IL-3, IL-4, IL-5,IL-6, IL-10, and IL-13. The invention also includes passive immunizationof patients with plasma or IgG from donors immunized with naturallyoccurring substances typically present in the human body which have beentreated to render them more immunogenic (e.g., IgE, histamine,leukotrienes, and their respective receptors), either with or withoutadministration of interferon gamma.

The present invention encompasses passive immunization by administeringplasma and/or IgG from donors immunized with an interleukin involved inIgE production (e.g., IL-3, IL-4, IL-5, IL-6, IL-10, and IL-13), incombination with plasma and/or IgG from a donor immunized with adifferent interleukin involved in IgE production.

Further, the invention encompasses administering plasma and/or IgG fromdonors immunized with at least one interleukin involved in IgEproduction (e.g., IL-3, IL-4, IL-5, IL-6, IL-10, and IL-13) which donorswere also immunized with at least one protein, or fragment thereof,selected from the group consisting of IgE, IgE receptor, an interleukinreceptor, an interferon-alpha, an interferon-alpha receptor, ahistamine, a histamine receptor, a leukotriene, and a leukotrienereceptor.

These methods of passive immunization not directly involving thegeneration of an immune response in a mammal may be particularly usefulto treat an IgE-mediated immune response where it is not possible togenerate an anti-interleukin immune response. Further, such methods arealso useful to treat immunosuppressed individuals who may be unable tomount a sufficient immune response to the interleukin involved in IgEproduction.

Thus, the present invention should be construed to encompass methods forinhibiting the effect(s) of an interleukin otherwise involved inproduction of IgE (e.g., IL-3, IL-4, IL-5, IL-6, IL-10, IL-13) such thatIgE production is inhibited or diminished by passive immunization byadministration of an anti-interleukin compound (e.g., an antibody to theinterleukin receptor, or a soluble receptor not associated with a cell)or by suppressing production of the interleukin or its receptor usingantisense nucleic acids.

With respect to passive immunization using antibodies, antibodies whichare directed against any of the aforementioned substances may beadministered to a mammal in order to reduce production of IgE in thepatient. The types of antibodies which may be used include polyclonalantibodies, monoclonal antibodies, phage-derived antibodies, syntheticantibodies, humanized antibodies, and the like. As noted herein,antibody technology is described, for example, in Harlow et al. (1999,supra). Polyclonal antibodies directed against an interleukin proteinmay be made by immunizing any suitable animal and obtaining immune serumfrom the animal at selected intervals following immunization.

Monoclonal antibodies directed against full length or peptide fragmentsof a protein of interest may be prepared using any well known monoclonalantibody preparation procedures, such as those described, for example,in Harlow et al. (supra). Quantities of the desired peptide may also besynthesized using chemical synthesis technology. Alternatively, DNAencoding the desired peptide may be cloned and expressed from anappropriate promoter/regulatory sequence in cells which are suitable forthe generation of large quantities of peptide. Monoclonal antibodiesdirected against the peptide are generated from mice immunized with thepeptide using standard procedures as referenced herein.

Nucleic acid encoding the monoclonal antibody obtained using theprocedures described herein may be cloned and sequenced using technologywhich is available in the art, and is described, for example, in Wrightet al. (1992, Critical Rev. Immunol. 12:125-168), and the referencescited therein. Further, the antibody may be “humanized” using thetechnology described in Wright et al., (supra) and in the referencescited therein.

To generate a phage antibody library, a cDNA library is first obtainedfrom mRNA which is isolated from cells, e.g., the hybridoma, whichexpress the desired protein to be expressed on the phage surface, e.g.,the desired antibody. cDNA copies of the mRNA are produced using reversetranscriptase. cDNA which specifies immunoglobulin fragments areobtained by PCR and the resulting DNA is cloned into a suitablebacteriophage vector to generate a bacteriophage DNA library comprisingDNA specifying immunoglobulin genes. The procedures for making abacteriophage library comprising heterologous DNA are well known in theart and are described, for example, in Sambrook et al. (1989, MolecularCloning. A Laboratory Manual, Cold Spring Harbor, N.Y.) and in Ausubelet al. (Ausubel et al., 1993, Current Protocols in Molecular Biology,Green & Wiley, New York).

Bacteriophage which encode the desired antibody may be engineered suchthat the protein is displayed on the surface thereof in such a mannerthat it is available for binding to its corresponding binding protein,e.g., the antigen against which the antibody is directed. Thus, whenbacteriophage which express a specific antibody are incubated in thepresence of a cell which expresses the corresponding antigen, thebacteriophage will bind to the cell. Bacteriophage which do not expressthe antibody will not bind to the cell. Such panning techniques are wellknown in the art and are described for example, in Wright et al.(supra).

Processes such as those described above, have been developed for theproduction of human antibodies using M13 bacteriophage display (Burtonet al., 1994, Adv. Immunol. 57:191-280). Essentially, a cDNA library isgenerated from mRNA obtained from a population of antibody-producingcells. The mRNA encodes rearranged immunoglobulin genes and thus, thecDNA encodes the same. Amplified cDNA is cloned into M13 expressionvectors creating a library of phage which express human Fab fragments ontheir surface. Phage which display the antibody of interest are selectedby antigen binding and are propagated in bacteria to produce solublehuman Fab immunoglobulin. Thus, in contrast to conventional monoclonalantibody synthesis, this procedure immortalizes DNA encoding humanimmunoglobulin rather than cells which express human immunoglobulin.

The procedures just presented describe the generation of phage whichencode the Fab portion of an antibody molecule. However, the inventionshould not be construed to be limited solely to the generation of phageencoding Fab antibodies. Rather, phage which encode single chainantibodies (scFv/phage antibody libraries) are also included in theinvention. Fab molecules comprise the entire Ig light chain, that is,they comprise both the variable and constant region of the light chain,but include only the variable region and first constant region domain(CH1) of the heavy chain. Single chain antibody molecules comprise asingle chain of protein comprising the Ig Fv fragment. An Ig Fv fragmentincludes only the variable regions of the heavy and light chains of theantibody, having no constant region contained therein. Phage librariescomprising scFv DNA may be generated following the procedures describedin Marks et al. (1991, J. Mol. Biol. 222:581-597). Panning of phage sogenerated for the isolation of a desired antibody is conducted in amanner similar to that described for phage libraries comprising Fab DNA.

The invention should also be construed to include synthetic phagedisplay libraries in which the heavy and light chain variable regionsmay be synthesized such that they include nearly all possiblespecificities (Barbas, 1995, Nature Medicine 1:837-839; de Kruif et al.1995, J. Mol. Biol. 248:97-105).

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art. The invention thusincludes an isolated DNA encoding an anti-interleukin antibody or DNAencoding a portion of the antibody.

To isolate DNA encoding an antibody, for example, DNA is extracted fromantibody expressing phage obtained as described herein. Such extractiontechniques are well known in the art and are described, for example, inSambrook et al. (supra) and in Ausubel et al. (supra).

Another form of antibody includes a nucleic acid sequence which encodesthe antibody and which is operably linked to promoter/regulatorysequences which can direct expression of the antibody in vivo. For adiscussion of this technology, see, for example, Cohen (1993, Science259:1691-1692), Fynan et al. (1993, Proc. Natl. Acad. Sci.90:11478-11482) and Wolff et al. (1991, Biotechniques 11:474-485) whichdescribe the use of naked DNA as an antibody/vaccine. For example, aplasmid containing suitable promoter/regulatory sequences operablylinked to a DNA sequence encoding an antibody may be directlyadministered to a patient using the technology described in theaforementioned references.

Alternatively, the promoter/enhancer sequence operably linked to DNAencoding the antibody may be contained within a vector, which vector isadministered to the patient. The vector may be a viral vector which issuitable as a delivery vehicle for delivery of the DNA encoding theantibody to the patient, or the vector may be a non-viral vector whichis suitable for the same purpose. Examples of viral and non-viralvectors for delivery of DNA to cells and tissues are well known in theart and are described, for example, in Ma et al. (1997, Proc. Natl.Acad. Sci. USA 94:12744-12746). Examples of viral vectors include, butare not limited to, a recombinant vaccinia virus, a recombinantadenovirus, a recombinant retrovirus, a recombinant adeno-associatedvirus, a recombinant avian poxvirus, and the like (Cranage et al., 1986,EMBO J. 5:3057-3063; International Patent Application No. WO94/17810,published Aug. 18, 1994; International Patent Application No.WO94/23744, published Oct. 27, 1994). Examples of non-viral vectorsinclude, but are not limited to, liposomes, polyamine derivatives ofDNA, and the like.

The identity, selection and means for obtaining a desired antibodyuseful for treatment or prevention of an allergic disease may beperformed by the skilled artisan using conventional technology when inpossession of the present invention.

Other anti-interleukin compounds may include, but are not limited to,isolated proteins and isolated peptides and isolated nucleic acidsequences encoding receptors for the desired interleukin, which whenadministered to the human, serve to block binding of the endogenousinterleukin to its cognate receptor on a cell, thereby inhibiting theaction of the endogenous interleukin. Isolated proteins and peptideshaving interleukin receptor activity, and isolated nucleic acidsencoding the same, may be chemically synthesized by conventional methodsknown in the art, or they may be purchased from a commercial source ifavailable. In one embodiment of the invention, the interleukin receptor,being a protein, a peptide or as used herein, a nucleic acid encodingthe same, may be produced using recombinant techniques in vitro insufficiently large quantities for use in a therapeutic composition foruse in treating or preventing an allergic disease. In addition, arecombinant virus vector comprising DNA encoding the desired interleukinreceptor may be prepared using conventional recombinant DNA technologyprocedures.

The interleukin receptor, or the antibody to the interleukin receptor ofthe invention (collectively referred to herein as an “anti-interleukincompound”), can be formulated in a pharmaceutical composition which issuitable for administration of the compound to a human patient. It willbe appreciated that the precise formulation and dosage amounts will varydepending upon any number of factors, including, but not limited to, thetype and severity of the disease to be treated, the route ofadministration, the age and overall health of the individual, the natureof the anti-interleukin compound, etc. However, the preparation of apharmaceutically acceptable composition having an appropriate pH,isotonicity, stability and other characteristics is within the skill ofthe art. Pharmaceutical compositions are described in the art, forexample, in Remington's Pharmaceutical Sciences (Genaro ed., 1985, MackPublishing Co., Easton, Pa.).

The amount of the anti-interleukin compound administered, whether it isadministered as protein or as nucleic acid, is sufficient to prevent,diminish or alleviate the allergic state. The pharmaceuticalcompositions useful for practicing the invention may be administered todeliver a dose of between about 1 ng/kg and about 100 mg/kg of patientbody weight. Suitable amounts of the anti-interleukin compound foradministration include doses which are high enough to have the desiredeffect without concomitant adverse effects. When the anti-interleukincompound is a protein or peptide, a preferred dosage range is from about10 to about 1000 μg of protein or peptide per kg of patient body weight.When the anti-interleukin compound is administered in the form of DNAencoding the same contained within a recombinant virus vector, a dosageof between about 10² and about 10¹¹ plaque forming units of virus per kgof patient body weight may be used. When naked DNA encoding theanti-interleukin compound is to be administered as the pharmaceuticalcomposition, a dosage of between about 10 μg about several mg of DNA perkg of patient body weight may be used.

In the practice of the methods of the invention, a compositioncontaining an anti-interleukin compound is administered to a patient ina sufficient amount to prevent, diminish or alleviate an asthmatic statein the individual. Patients to be treated include children and adultswho have atopic allergy. In particular, patients to be treated includethose who have atopic asthma.

The frequency of administration of an anti-interleukin compound to apatient will also vary depending on several factors including, but notlimited to, the type and severity of the allergy to be treated, theroute of administration, the age and overall health of the individual,the nature of the anti-interleukin compound, etc. It is contemplatedthat the frequency of administration of the anti-interleukin compound tothe patient may vary from about once every few months to about once amonth, to about once a week, to about once per day, to about severaltimes daily.

Pharmaceutical compositions that are useful in the methods of theinvention may be administered systemically in parenteral, oral solid andliquid formulations, ophthalmic, suppository, aerosol, topical or othersimilar formulations. In addition to the appropriate anti-interleukincompound, these pharmaceutical compositions may containpharmaceutically-acceptable carriers and other ingredients known toenhance and facilitate drug administration. Thus, such compositions mayoptionally contain other components, such as adjuvants, e.g., aqueoussuspensions of aluminum and magnesium hydroxides, and/or otherpharmaceutically acceptable carriers, such as saline. Other possibleformulations, such as nanoparticles, liposomes, resealed erythrocytes,and immunologically based systems may also be used to administer theappropriate anti-interleukin compound to a patient according to themethods of the invention. Oral delivery of antibodies is described inReilly et al. (1997, Clin. Pharmacol. 32:313-323).

In the case of treatment of asthma, the composition of the invention ispreferably administered to the human by a lung inhalation route, i.e.,via a nebulizer or other lung inhalation device.

An anti-interleukin compound may be administered in conjunction withother compounds which are used to treat asthma. Such compounds include,but are not limited to, corticosteroids, sodium cromolyn,methylxanthines, leukotriene pathway modifiers, anti-cholinergic agents,and rapid relief medications that counteract bronchospasm, e.g.,primarily beta-adrenergic agents. The choice of which additionalcompound to administer will vary depending upon any number of the sametypes of factors that govern the selection of dosage and administrationfrequency of the anti-interleukin compound. Selection of these types ofcompounds for use in conjunction with an anti-interleukin compound forpractice of the method of the invention is well within the skill ofthose in the art.

The invention encompasses use of interferon gamma as both a therapeuticagent to treat asthma and as a preventative agent to prevent asthmaattacks. Thus, interferon gamma can be used in both instances.Interferon gamma can be administered in doses ranging from about 10⁴ to10⁶ units per square meter of body surface area. Further, interferongamma is administered before, along with, or after administration ofIgE, IgE receptor, interleukin, interleukin receptor, interferon-alpha,interferon-alpha receptor, histamine, histamine receptor, leukotriene,leukotriene receptor (i.e., active immunization) and/or antibodies toany or all of the preceding substances excluding anti-interleukinantibodies (i.e., passive immunization). The invention encompassesadministration of any of these substances alone, together, or in anycombination thereof, either with or without interferon gamma.

Antisense Molecules

The invention also includes a method of preventing an allergic responsein a human comprising administering an antisense nucleic acidcomplementary to a nucleic acid encoding at least one of IgE, aninterleukin involved in IgE production such as IL-3, IL-4, IL-5, IL-6,IL-10, and IL-13, an interferon-alpha, a histamine, and a leukotriene.One skilled in the art would appreciate, based upon the disclosureprovided herein, that inhibiting expression of a nucleic acid encoding asubstance involved in IgE production causes a reduction in theproduction of IgE which, in turn, prevents an allergic response. In thisregard, certain molecules, including antisense nucleic acids andribozymes, are useful in inhibiting expression of a nucleic acidcomplementary thereto.

Similarly, one skilled in the art would appreciate, based upon thedisclosure provided herein, that the administration of an antisensenucleic acid complementary to a nucleic acid encoding a receptor forIgE, an interleukin involved in IgE production such as IL-3, IL-4, IL-5,IL-6, IL-10, and IL-13, an interferon-alpha, a histamine, and aleukotriene would also inhibit IgE production thereby treating and/orpreventing an allergic immune response. That is, inhibition ofproduction of a receptor prevents the ligand/receptor interaction(s)required for induction of IgE synthesis from taking place. Thus,inhibition of expression of a receptor involved in induction of IgEproduction reduces IgE biosynthesis and prevents and/or treats anallergic response mediated by IgE.

In one aspect, the invention includes a method of inhibiting theproduction of at least one of IgE, an interleukin involved in IgEproduction, an interferon-alpha, a histamine, and a leukotriene byadministering to a mammal an antisense nucleic acid complementary to anucleic acid encoding the protein. Thus, by inhibiting the expression ofthe protein which would otherwise be involved in increasing productionof IgE, the antisense nucleic acid inhibits the production of IgE andalso treats and/or prevents an allergic response.

Additionally, the invention includes a method of inhibiting theproduction of IgE by administering to a mammal an antisense nucleic acidcomplementary to a nucleic acid encoding a receptor for IgE, aninterleukin involved in IgE production, an interferon-alpha, ahistamine, and a leukotriene. Therefore, the invention encompassesinhibiting the expression of a receptor involved in IgE productionthereby inhibiting the requisite ligand/receptor interaction(s)necessary for the induction of IgE production. As disclosed previouslyelsewhere herein, inhibition of IgE production prevents and/or treats anallergic response driven by IgE production such as, but not limited to,asthma.

Antisense molecules and their use for inhibiting gene expression arewell known in the art (see, e.g., Cohen, 1989, In:Oligodeoxyribonucleotides, Antisense Inhibitors of Gene Expression, CRCPress). Antisense nucleic acids are DNA or RNA molecules that arecomplementary, as that term is defined elsewhere herein, to at least aportion of a specific mRNA molecule (Weintraub, 1990, ScientificAmerican 262:40). In the cell, antisense nucleic acids hybridize to thecorresponding mRNA, forming a double-stranded molecule therebyinhibiting the translation of genes.

The use of antisense methods to inhibit the translation of genes isknown in the art, and is described, for example, in Marcus-Sakura, 1988,Anal. Biochem. 172:289. Such antisense molecules may be provided to thecell via genetic expression using DNA encoding the antisense molecule astaught by Inoue, 1993, U.S. Pat. No. 5,190,931 (incorporated byreference herein in its entirety).

Alternatively, antisense molecules of the invention may be madesynthetically and then provided to a human or veterinary patient.Antisense oligomers of about 15 nucleotides are preferred, since theyare easily synthesized and introduced into a target cell. Syntheticantisense molecules contemplated by the invention includeoligonucleotide derivatives known in the art which have improvedbiological activity compared to unmodified oligonucleotides (see Cohen,supra; Tullis, 1991, U.S. Pat. No. 5,023,243, incorporated by referenceherein in its entirety).

In addition to using an antisense molecule to inhibit expression of anucleic acid encoding an interleukin or an interleukin receptor, thepresent invention encompasses the use of ribozymes in this manner.Ribozymes are another nucleic acid that may be transfected into a cellto inhibit nucleic acid expression in the cell. Ribozymes and their usefor inhibiting gene expression are also well known in the art (see,e.g., Cech et al., 1992, J. Biol. Chem. 267:17479-17482; Hampel et al.,1989, Biochemistry 28:4929-4933; Eckstein et al., InternationalPublication No. WO 92/07065; Altman et al., U.S. Pat. No. 5,168,053,incorporated by reference herein in its entirety). Ribozymes are RNAmolecules possessing the ability to specifically cleave othersingle-stranded RNA in a manner analogous to DNA restrictionendonucleases. Through the modification of nucleotide sequences encodingthese RNAs, molecules can be engineered to recognize specific nucleotidesequences in an RNA molecule and cleave it (Cech, 1988, J. Amer. Med.Assn. 260:3030). A major advantage of this approach is that, becausethey are sequence-specific, only mRNAs with particular sequences areinactivated.

There are two basic types of ribozymes, namely, tetrahymena-type(Hasselhoff, 1988, Nature 334:585) and hammerhead-type. Tetrahymena-typeribozymes recognize sequences which are four bases in length, whilehammerhead-type ribozymes recognize base sequences 11-18 bases inlength. The longer the sequence, the greater the likelihood that thesequence will occur exclusively in the target mRNA species.Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes for inactivating specific mRNA species, and18-base recognition sequences are preferable to shorter recognitionsequences which may occur randomly within various unrelated mRNAmolecules.

Ribozymes useful for inhibiting the expression of the proteins ofinterest may be designed by incorporating target sequences into thebasic ribozyme structure which are complementary to the mRNA sequence ofthe nucleic acid encoding the protein of interest. Ribozymes targetingan interleukin involved in IgE production may be synthesized usingcommercially available reagents (Applied Biosystems, Inc., Foster City,Calif.) or they may be expressed from DNA encoding them.

Compounds that inhibit expression of a nucleic acid encoding aninterleukin involved in IgE production and/or encoding its receptor,i.e., antisense and ribozyme molecules, are administered to a human asdescribed elsewhere herein for the delivery of nucleic acids generally.

In addition, the instant invention includes methods of treating orpreventing asthma, allergy, and autoimmune conditions comprisingadministering interferon-gamma (IFNγ).

Active and Passive Methods Combined

One skilled in the art would appreciate, based upon the disclosureprovided herein, that the above-disclosed allergy methods of active andpassive immunization can be combined to effect treatment and/orprevention of asthma, and allergic and autoimmune conditions. Thus, theinvention encompasses active/passive immunization with at least one of:an interleukin involved in IgE production, a receptor of such aninterleukin, an anti-interleukin receptor antibody, an interferon-alpha,an interferon-alpha receptor, an anti-interferon-alpha antibody, ananti-interferon alpha receptor antibody, a soluble interferon-alphareceptor, histamine, histamine receptor, anti-histamine antibody,anti-histamine receptor antibody, leukotriene, leukotriene receptor,anti-leukotriene antibody, anti-leukotriene receptor antibody, IgE, IgEreceptor, anti-IgE antibody, and anti-IgE receptor antibody, and anycombination of these substances.

This is because as disclosed herein, any decrease in the interactionbetween IgE and its receptor and/or any decrease in the interaction ofthe substances released by cells activated by binding of IgE (e.g.,histamine, leukotrienes) with their receptors (e.g., histamine receptorand leukotriene receptor) alleviates, prevents, or treats asthma, andother allergic and autoimmune conditions mediated by IgE and substancesproduced upon stimulation by IgE. Therefore, methods that inhibit therelease of IgE and/or its action, including methods that inhibit, amongother things, IgE binding with its receptor, binding of variousinterleukins with their receptors, binding of interferon-alpha with itscognate receptor, and interaction of histamine and leukotrienes withtheir respective receptors, all prevent, treat, or both, asthma,allergies, and autoimmune conditions mediated by such interactions.

In addition, the various methods, both passive and active immunizationusing proteins or nucleic acid encoding protein, can be combined withmethods of treating or preventing asthma, allergy, and autoimmuneconditions comprising administering interferon gamma.

Further, with regard to passive immunization by administering anantibody that specifically binds with an interleukin involved in IgEproduction, the antibody to the interleukin is not administered alone orin combination with another antibody to the same or a differentinterleukin involved in IgE production. Instead, with regard to passiveimmunization using antibodies directed to interleukins involved in IgEproduction, an antibody to an interleukin involved in IgE production isadministered in combination with at least one other protein selectedfrom the following group: a soluble receptor of such an interleukin, ananti-interleukin receptor antibody, an interferon-alpha receptor, ananti-interferon-alpha antibody, an anti-interferon alpha receptorantibody, an anti-histamine antibody, an anti-histamine receptorantibody, a histamine receptor, a leukotriene receptor, ananti-leukotriene antibody, anti-leukotriene receptor antibody, an IgEreceptor, an anti-IgE antibody, and anti-IgE receptor antibody, and anycombination of these substances.

Kits

The invention includes various kits comprising at least one substance(e.g., IgE, an interleukin involved in IgE production, aninterferon-alpha, a histamine, a leukotriene, and their respectivereceptors) that when administered either as a protein or as a nucleicacid encoding the protein, generate an immune response such that thenecessary ligand/receptor interaction is blocked and IgE production isinhibited thereby. The kits further comprise an applicator andinstructional materials which describe use of the compound to performthe methods of the invention

The invention also includes various kits comprising at least onesubstance (e.g., an anti-IgE antibody, a soluble IgE receptor, ananti-IgE receptor antibody, a soluble receptor for an interleukininvolved in IgE production, an anti-interleukin receptor antibody, ananti-interferon-alpha antibody, an anti-interferon-alpha receptorantibody, a soluble interferon-alpha receptor, an anti-histamineantibody, a soluble histamine receptor, an anti-histamine receptorantibody, an anti-leukotriene antibody, a soluble leukotriene receptor,and an anti-leukotriene receptor antibody) that inhibits production ofIgE when administered to a mammal without the need to generate an immuneresponse in the animal although an immune response may be generated.

The invention encompasses kits comprising at least one antisense nucleicacid complementary to a nucleic acid encoding at least one IgE, IgEreceptor, interleukin involved in IgE production, a receptor of such aninterleukin, an interferon-alpha, an interferon-alpha receptor, ahistamine, a histamine receptor, a leukotriene, and a leukotrienereceptor. Administration of such an antisense nucleic acid inhibitsexpression of the protein thereby inhibiting the ligand/receptorinteraction required for IgE production in which the protein wouldotherwise participate. Inhibiting the requisite ligand/receptorinteraction, in turn, inhibits production of IgE thereby inhibitingallergic responses mediated thereby.

The kits further comprise and applicator and instructional materialswhich describe use of the compound to perform the methods of theinvention. Although exemplary kits are described below, the contents ofother useful kits will be apparent to the skilled artisan in light ofthe present disclosure. Each of these kits is included within theinvention.

In one aspect, the invention includes a kit for preventing an allergicresponse in a mammal. The kit is used pursuant to the methods disclosedin the invention. Briefly, the kit may be used to administer an allergyvaccine comprising at least one of IgE, an interleukin involved in theproduction of IgE, an interferon-alpha, a histamine, a leukotriene, anda receptor of these proteins. The allergy vaccine may be introduced as aprotein, or fragment thereof, or as an isolated nucleic acid encoding aprotein, or portion thereof, into a mammal in order to decrease thelevel of the interleukin in the mammal thereby reducing the level of IgEin the mammal which, in turn, prevents the allergic response in themammal.

The kit further comprises an applicator useful for administering theprotein and/or nucleic acid encoding the protein to the mammal. Theparticular applicator included in the kit will depend on the method usedto administer the protein and/or the nucleic acid encoding the same tothe mammal, and such applicators are well-known in the art and mayinclude, among other things, a pipette, a syringe, a dropper, a spray,an inhaler, a nebulizer, an endotracheal tube, a bronchoscope, and thelike. Moreover, the kit comprises an instructional material for the useof the kit. These instructions simply embody the disclosure providedherein.

In another aspect, the kit includes interferon gamma. The interferongamma is administered pursuant to the methods disclosed herein. Briefly,interferon gamma is known to inhibit the production and/or effects ofIL-4 including the production of IgE. Therefore, interferon gammaeffects a further reduction in IgE level thereby preventing an allergicresponse in the human.

The invention includes a kit for treating an allergy in a mammal. Thekit is used pursuant to the methods disclosed in the invention. Briefly,the kit comprises and may be used to administer at least one of asoluble IgE receptor, an anti-IgE receptor antibody, a soluble receptorfor an interleukin involved in IgE production (e.g., IL-3, IL-4, IL-5,IL-6, IL-10, and IL-13), an anti-interleukin receptor antibody, ananti-interferon-alpha antibody, an anti-interferon-alpha receptorantibody, a soluble interferon-alpha receptor, an anti-histamineantibody, a soluble histamine receptor, an anti-histamine receptorantibody, an anti-leukotriene antibody, a soluble leukotriene receptor,and an anti-leukotriene receptor antibody. The substance may beadministered to the mammal as a protein, or fragment thereof, or as anisolated nucleic acid encoding a protein, or portion thereof, in orderto block the ligand/receptor interactions required for IgE productionthereby reducing the level of IgE produced in the mammal which, in turn,prevents the allergic response in the mammal.

The kit further comprises an applicator useful for administering theprotein and/or nucleic acid encoding the protein to the mammal. Theparticular applicator included in the kit will depend on the method usedto administer the interleukin and/or the nucleic acid encoding same tothe mammal and such applicators are well-known in the art and mayinclude, among other things, a pipette, a syringe, a dropper, a spray,an inhaler, a nebulizer, an endotracheal tube, a bronchoscope, and thelike. Moreover, the kit comprises an instructional material for the useof the kit. These instructions simply embody the disclosure providedherein.

In another aspect, the kit includes interferon gamma. The interferongamma is administered pursuant to the methods disclosed herein. Briefly,interferon gamma is known to inhibit the production and/or effects ofIL-4 including the production of IgE. Therefore, interferon gammaeffects a further reduction in IgE level thereby preventing an allergicresponse in the human.

The invention includes a kit for treating an allergy in a mammal. Thekit is used pursuant to the methods disclosed in the invention. Briefly,the kit comprises and may be used to administer at least oneanti-interleukin antibody where the interleukin is involved in IgEproduction (e.g., IL-3, IL-4, IL-5, IL-6, IL-10, and IL-13) incombination with a second protein selected from the group consisting ofa soluble IgE receptor, an anti-IgE receptor antibody, a solublereceptor for an interleukin involved in IgE production (e.g., IL-3,IL-4, IL-5, IL-6, IL-10, and IL-13), an anti-interleukin receptorantibody, an anti-interferon-alpha antibody, an anti-interferon-alphareceptor antibody, a soluble interferon-alpha receptor, ananti-histamine antibody, a soluble histamine receptor, an anti-histaminereceptor antibody, an anti-leukotriene antibody, a soluble leukotrienereceptor, and an anti-leukotriene receptor antibody. The proteins can beadministered to the mammal as a protein, or fragment thereof, or as anisolated nucleic acid encoding a protein, or portion thereof, in orderto block the ligand/receptor interactions required for IgE productionthereby reducing the level of IgE produced in the mammal which, in turn,prevents the allergic response in the mammal.

The kit further comprises an applicator useful for administering theprotein and/or nucleic acid encoding the protein to the mammal. Theparticular applicator included in the kit will depend on the method usedto administer the interleukin and/or the nucleic acid encoding same tothe mammal and such applicators are well-known in the art and mayinclude, among other things, a pipette, a syringe, a dropper, a spray,an inhaler, a nebulizer, an endotracheal tube, a bronchoscope, and thelike. Moreover, the kit comprises an instructional material for the useof the kit. These instructions simply embody the disclosure providedherein.

In another aspect, the kit includes interferon gamma. The interferongamma is administered pursuant to the methods disclosed herein. Briefly,interferon gamma is known to inhibit the production and/or effects ofIL-4 including the production of IgE. Therefore, interferon gammaeffects a further reduction in IgE level thereby preventing an allergicresponse in the human.

The invention further includes a kit for treating an allergy in amammal. The kit is used pursuant to the methods disclosed in theinvention. Briefly, the kit comprises and may be used to administer anantisense nucleic acid complementary to a nucleic acid encoding IgE, IgEreceptor, an interleukin involved in the production of IgE (e.g., IL-3,IL-4, IL-5, IL-6, IL-10, and IL-13), an interleukin receptor, aninterferon-alpha, an interferon-alpha receptor, a histamine, a histaminereceptor, a leukotriene, and a leukotriene receptor. The antisensenucleic acid inhibits expression of nucleic acid to which it iscomplementary thereby inhibiting production of the protein encodedthereby. The reduction in expression of the protein in turn inhibits theligand/receptor interactions necessary for production of IgE bydecreasing the amount of protein available to participate in suchinteractions. Decreasing the level of IgE in the mammal, in turn, treatsthe allergy in the mammal.

The kit further comprises an applicator useful for administering theanti-sense nucleic acid to the mammal pursuant to the methods disclosedelsewhere herein. The particular applicator included in the kit willdepend on the method used to administer the antisense nucleic acid tothe mammal and such applicators are well-known in the art and mayinclude, among other things, a pipette, a syringe, a dropper, anebulizer, an endotracheal tube, a bronchoscope, and the like. Moreover,the kit comprises an instructional material for the use of the kit.These instructions simply embody the disclosure provided herein.

In one aspect, the kit comprises interferon gamma which is administeredpursuant to the methods disclosed elsewhere herein.

The invention encompasses a kit for preventing an allergic response in amammal. The kit is used pursuant to the methods disclosed in theinvention. Briefly, the kit can be used to administer a pharmaceuticalcomposition where the composition comprises at least one allergy vaccinein an amount effective for preventing an allergic response in a mammal.The pharmaceutical composition also comprises a pharmaceuticallyacceptable carrier.

As stated previously elsewhere herein, the allergy vaccine comprises aprotein selected from the group consisting of IgE, an IgE receptor, aninterleukin involved in the production of IgE, a receptor for aninterleukin involved in the production of IgE, an interferon-alpha, aninterferon-alpha receptor, a histamine, a histamine receptor, aleukotriene, and a leukotriene receptor.

The kit further encompasses an applicator, and an instructional materialfor the use thereof in accordance with the methods disclosed elsewhereherein.

The allergy vaccine comprised by the pharmaceutical composition can beintroduced as a protein, peptide, or a fragment thereof, or as anisolated nucleic acid encoding a protein, peptide or fragment thereof,in order to inhibit the ligand/receptor interactions that mediate IgEproduction thereby inhibiting production of IgE thus preventing anallergic response mediated by IgE.

In one aspect, the kit further comprises interferon gamma.

The invention further includes a kit for treating an allergy in amammal. The kit comprises a pharmaceutical composition comprising atleast one protein in an amount effective for treating an allergy in amammal. The pharmaceutical composition further comprises apharmaceutically acceptable carrier.

The protein is selected from an anti-IgE antibody, an anti-IgE receptorantibody, a soluble IgE receptor, an anti-interleukin receptor antibody,a soluble interleukin receptor, an anti-interferon-alpha antibody, ananti-interferon-alpha receptor antibody, a soluble interferon-alphareceptor, an anti-histamine antibody, an anti-histamine receptorantibody, a soluble histamine receptor, an anti-leukotriene antibody, ananti-leukotriene receptor antibody, and a soluble leukotriene receptor,wherein the interleukin is at least one interleukin involved in theproduction of IgE in a mammal.

The kit comprises an applicator, and an instructional material for theuse of the kit in accordance with the methods disclosed elsewhereherein.

In one aspect, the kit further comprises interferon gamma.

The invention includes a kit for treating an allergy in a mammal. Thekit comprises a pharmaceutical composition comprising an antisensenucleic acid in an amount effective for treating an allergy in a mammal.The antisense nucleic acid is complementary to a nucleic acid encodingat least one ingredient selected from the group consisting of IgE, anIgE receptor, an interleukin involved in the production of IgE in amammal, a receptor to an interleukin involved in the production of IgEin a mammal, an interferon-alpha, an interferon-alpha receptor, ahistamine, a histamine receptor, a leukotriene, and a leukotrienereceptor. Moreover, the pharmaceutical composition further comprises apharmaceutically acceptable carrier.

The kit comprises an applicator, and an instructional material for theuse thereof to inhibit production of IgE thereby treating an allergy ina mammal.

In another aspect, the kit further comprises interferon gamma.

The invention includes a pharmaceutical composition comprising at leastone protein selected from the group consisting of an IgE, an IgEreceptor, an interleukin involved in production of IgE in a mammal, areceptor for an interleukin involved in production of IgE in a mammal,an interferon-alpha, an interferon-alpha receptor, a histamine, ahistamine receptor, a leukotriene, a leukotriene receptor. The proteinis in an in an amount effective for preventing an allergic response in amammal. Such amount can be readily determined based upon the teachingsdisclosed herein. Further, the composition comprises apharmaceutically-acceptable carrier.

The invention includes a pharmaceutical composition comprising at leastone isolated nucleic acid encoding a protein selected from the groupconsisting of an IgE, an IgE receptor, an interleukin involved inproduction of IgE in a mammal, a receptor for an interleukin involved inproduction of IgE in a mammal, an interferon-alpha, an interferon-alphareceptor, a histamine, a histamine receptor, a leukotriene, aleukotriene receptor. The nucleic acid is present in an amount effectivefor preventing an allergic response in a mammal. Such amount can bereadily determined based upon the teachings disclosed herein. Further,the composition comprises a pharmaceutically-acceptable carrier.

The invention further includes a pharmaceutical composition comprisingat least one protein in an amount effective for treating an allergy in amammal. The protein is selected from the group consisting of an anti-IgEantibody, an anti-IgE receptor antibody, a soluble IgE receptor, ananti-interleukin receptor antibody, a soluble interleukin receptor, ananti-interferon-alpha antibody, an anti-interferon-alpha receptorantibody, a soluble interferon-alpha receptor, an anti-histamineantibody, an anti-histamine receptor antibody, a soluble histaminereceptor, an anti-leukotriene antibody, an anti-leukotriene receptorantibody, and a soluble leukotriene receptor. The interleukin receptoris selected from the group consisting a receptor for an interleukininvolved in the production of IgE in a mammal (e.g., IL-3, IL-4, IL-5,IL-6, IL-10, and IL-13). The composition further comprises apharmaceutically acceptable carrier.

The invention further includes a pharmaceutical composition comprisingat least one antibody that specifically binds to at least oneinterleukin involved in IgE production (i.e., IL-3, IL-4, IL-5, IL-6,IL-10, and IL-13) and a second protein in an amount effective fortreating an allergy in a mammal selected from the group consisting of ananti-IgE antibody, an anti-IgE receptor antibody, a soluble IgEreceptor, an anti-interleukin receptor antibody, a soluble interleukinreceptor, an anti-interferon-alpha antibody, an anti-interferon-alphareceptor antibody, a soluble interferon-alpha receptor, ananti-histamine antibody, an anti-histamine receptor antibody, a solublehistamine receptor, an anti-leukotriene antibody, an anti-leukotrienereceptor antibody, and a soluble leukotriene receptor. The interleukinreceptor is selected from the group consisting a receptor for aninterleukin involved in the production of IgE in a mammal (e.g., IL-3,IL-4, IL-5, IL-6, IL-10, and IL-13). The composition further comprises apharmaceutically acceptable carrier.

The invention includes that the above-disclosed pharmaceuticalcompositions each can comprise interferon gamma.

Definitions

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“Alleviate,” as the term is used herein, means reducing the severity ofthe symptoms of the disease or disorder.

“Allergy vaccine,” as the term is used herein, means an immunogenicsubstance which when administered to a human or veterinary patient,induces a detectable immune response to the substance thereby mediatinga decrease in the amount of circulating IgE or in the level of IL-4 orIL-5 compared to the level of circulating IgE or in the level of IL-4 orIL-5 in the human or veterinary patient prior to the administration ofthe immunogenic substance or when compared to the level of circulatingIgE or the level of IL-4 or IL-5 in an otherwise identical human orveterinary patient to which the immunogenic substance is notadministered. Such immunogenic substance includes IgE, an IgE receptor,an interleukin involved in production of IgE in a mammal, a receptor foran interleukin involved in production of IgE in a mammal, histamine, ahistamine receptor, a leukotriene, a leukotriene receptor, and anycombination thereof.

An “amount effective for” treating an allergy and/or preventing anallergic response, as the term is used herein, means a quantity of aprotein, peptide, and fragment thereof, or nucleic acid encoding theprotein, peptide, and fragment thereof, and/or an antisense nucleic acidcomplementary to such nucleic acid, which when administered to a mammal,inhibits the production of IgE or reduces the level of IgE in a mammal.Further, as stated previously elsewhere herein, the amount of IgEproduced and/or present in a mammal can be easily assessed using a widevariety of methods well-known in the art.

The term “antibody”, as used herein, refers to an immunoglobulinmolecule which is able to specifically bind to a specific epitope on anantigen.

Antibodies can be intact immunoglobulins derived from natural sources orfrom recombinant sources and can be immunoreactive portions of intactimmunoglobulins.

Antibodies are typically tetramers of immunoglobulin molecules. Theantibodies in the present invention may exist in a variety of formsincluding, for example, polyclonal antibodies and humanized antibodies(Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, ColdSpring Harbor, N.Y.; Harlow et al., 1988, In: Antibodies: A LaboratoryManual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl.Acad. Sci. U.S.A. 85:5879-5883; Bird et al., 1988, Science 242:423-426).

By the term “specifically binds”, as used herein, is meant, for example,an antibody which recognizes and binds an interleukin involved in IgEproduction, but does not substantially recognize or bind other moleculesin a sample.

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

As used herein, the term “antisense nucleic acid” means a nucleic acidpolymer, at least a portion of which is complementary to another nucleicacid. The antisense nucleic acid may comprise between about fourteen andabout fifty or more nucleotides. Preferably, the antisense nucleic acidcomprises between about twelve and about thirty nucleotides. Morepreferably, the antisense nucleic acid comprises between about sixteenand about twenty-one nucleotides. The antisense nucleic acid mayinclude, but is not limited to, phosphorothioate oligonucleotides andother modifications of oligonucleotides. Methods for synthesizingoligonucleotides, phosphorothioate oligonucleotides, and otherwisemodified oligonucleotides are well known in the art (U.S. Pat. No.5,034,506; Nielson et al., 1991, Science 254:1497).

The term “antisense” refers particularly to the nucleic acid sequence ofthe non-coding strand of a double stranded DNA molecule or, in the caseof some viruses, a single or double stranded RNA molecule, encoding aprotein, or to a sequence which is substantially homologous to thenon-coding strand. It is not necessary that the antisense sequence becomplementary solely to the coding portion of the coding strand of thenucleic acid molecule. The antisense sequence may be complementary toregulatory sequences specified on the coding strand of a nucleic acidmolecule encoding a protein, which regulatory sequences controlexpression of the coding sequences.

The term “sense”, as used herein, refers to the nucleic acid sequence ofthe single or double-stranded nucleic acid molecule which encodes aprotein, or a sequence which is substantially homologous to that strand.However, the nucleic acid sequence is not limited solely to the portionof the coding strand encoding a protein; rather, the sequence mayinclude regulatory sequences involves in, for example, the control ofexpression of the coding sequence.

By the term “applicator” as the term is used herein, is meant any deviceincluding, but not limited to, a hypodermic syringe, a pipette, adropper, and the like, for administering the isolated nucleic acid,polypeptide or antibody to a cell or to an animal.

By the terms “coding” and “encoding,” as these terms are used herein, ismeant that the nucleotide sequence of a nucleic acid is capable ofspecifying a particular polypeptide of interest. That is, the nucleicacid may be transcribed and/or translated to produce the polypeptide.Thus, for example, an isolated nucleic acid encoding an interleukin iscapable of being transcribed and/or translated to produce an interleukinpolypeptide. Similarly, an isolated nucleic acid encoding ananti-interleukin compound is capable of being transcribed and/ortranslated to produce an anti-interleukin polypeptide, e.g., aninterleukin receptor.

“Complementary” as used herein refers to the broad concept of subunitsequence complementary between two nucleic acids, e.g., two DNAmolecules. When a nucleotide position in both of the molecules isoccupied by nucleotides normally capable of base pairing with eachother, then the nucleic acids are considered to be complementary to eachother at this position. Thus, two nucleic acids are complementary toeach other when a substantial number (at least 50%) of correspondingpositions in each of the molecules are occupied by nucleotides whichnormally base pair with each other (e.g., A:T and G:C nucleotide pairs).

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate.

In contrast, a “disorder” in an animal is a state of health in which theanimal is able to maintain homeostasis, but in which the animal's stateof health is less favorable than it would be in the absence of thedisorder. Left untreated, a disorder does not necessarily cause afurther decrease in the animal's state of health.

By the term “immunogenic interleukin” as used herein, is meant aninterleukin which when introduced into a human elicits at least one of ahumoral or a cellular immune response which is specifically directed(i.e., would not be elicited but for the presence of the introducedinterleukin) against the immunogenic interleukin.

Further, the term “immunogenic interleukin” should be construed toencompass not only polypeptide molecules, or fragments thereof, butshould also be construed to encompass a nucleic acid encoding theinterleukin or its receptor, whether as naked DNA or comprising avector. The term should also be construed to encompass a cell expressingthe interleukin of interest and/or an interleukin receptor such that theimmunogenic interleukin is presented to the immune system so that adetectable immune response, whether humoral and/or cellular, to theinterleukin and/or to an interleukin receptor is generated.

The term “interferon” or “IFN,” as used interchangeably herein, refersto any know subtype of interferon. For example, “interferon-alpha”includes any of the fifteen known subtypes of interferon-alpha (IFNα),or any that may be determined in the future.

An “isolated nucleic acid” refers to a nucleic acid segment or fragmentwhich has been separated from sequences which flank it in a naturallyoccurring state, e.g., a DNA fragment which has been removed from thesequences which are normally adjacent to the fragment, e.g., thesequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, e.g., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g.,as a cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

The use of the terms “nucleic acid encoding” or “nucleic acid coding”should be construed to include the RNA or DNA sequence which encodes thedesired protein and any necessary 5′ or 3′ untranslated regionsaccompanying the actual coding sequence.

As used herein, the term “pharmaceutically-acceptable carrier” means achemical composition with which an appropriate anti-interleukin compoundmay be combined and which, following the combination, can be used toadminister the anti-interleukin compound to a patient.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulator sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

By describing two polynucleotides as “operably linked” is meant that asingle-stranded or double-stranded nucleic acid moiety comprises the twopolynucleotides arranged within the nucleic acid moiety in such a mannerthat at least one of the two polynucleotides is able to exert aphysiological effect by which it is characterized upon the other. By wayof example, a promoter operably linked to the coding region of a gene isable to promote transcription of the coding region.

A “receptor” is a protein found on the surface of a cell or in itscytoplasm, that has a binding site with high affinity to a particularsubstance (e.g., a cytokine, a hormone, a neurotransmitter, and thelike), referred to as the receptor's “ligand.” By competitivelyinhibiting the ability of the receptor to bind its cognate ligand, suchas by using, for example, an antibody to the ligand, an antibody to thereceptor, or an analog of the receptor (e.g. a soluble receptor notassociated with a cell or a cell surface), a receptor/ligand interactionrequired for induction of IgE production is inhibited thereby inhibitingor reducing the production of IgE.

By the term “scFv/phage” are used herein, is meant a phage particlewhich expresses the Fv portion of an antibody as a single chain.

As used herein, to “treat” means reducing the frequency with whichsymptoms of the allergy disease are experienced by a patient.

By the term “vaccine,” as the term is used herein, is meant a protein,or fragment thereof, which when administered to a human or veterinarypatient, induces a detectable immune response, humoral and/or cellular,to at least one protein or a fragment thereof.

By the term “vector” as used herein, is meant any plasmid or virusencoding an exogenous nucleic acid. The term should also be construed toinclude non-plasmid and non-viral compounds which facilitate transfer ofnucleic acid into virions or cells, such as, for example, polylysinecompounds and the like. The vector may be a viral vector which issuitable as a delivery vehicle for delivery of the interleukin proteinor nucleic acid encoding the interleukin, to the patient, or the vectormay be a non-viral vector which is suitable for the same purpose.Examples of viral and non-viral vectors for delivery of DNA to cells andtissues are well known in the art and are described, for example, in Maet al. (1997, Proc. Natl. Acad. Sci. U.S.A. 94:12744-12746). Examples ofviral vectors include, but are not limited to, a recombinant vacciniavirus, a recombinant adenovirus, a recombinant retrovirus, a recombinantadeno-associated virus, a recombinant avian pox virus, and the like(Cranage et al., 1986, EMBO J. 5:3057-3063; International PatentApplication No. WO94/17810, published Aug. 18, 1994; InternationalPatent Application No. WO94/23744, published Oct. 27, 1994). Examples ofnon-viral vectors include, but are not limited to, liposomes, polyaminederivatives of DNA, and the like.

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Sample protocols which employ the methods of the invention are nowdisclosed.

The patient is immunized with at least one appropriate interleukin, orcombination thereof, either co-administered or wherein one interleukinis administered before the other. The interleukin (either as protein ornucleic acid encoding same) is administered parenterally (subcutaneouslyor intramuscularly) in doses of approximately 0.01 to about 1 mg/kg.

Immune plasma is collected from the patient immunized as disclosed aboveand administered parenterally to a patient suffering from an allergy.The immune plasma is used to confer passive immunity to theinterleukin(s) of interest to patients suffering from an allergy. Morespecifically, the plasma is administered to a patient suffering fromasthma.

Alternatively, anti-interleukin antibodies are purified from the immuneplasma before being used to confer passive immunity to the interleukin.

Anti-interleukin antibody is administered intramuscularly (IM) orintravenously (IV) in doses of approximately 0.1 to about 100 mg/kg.

Anti-interleukin antibody is administered as disclosed above, and then,about 12 hours to about 96 hours after this, gamma interferon isadministered IM in doses of about 1 to about 3×10⁶ IU/kg body weight.

An interleukin receptor and/or an antibody to an interleukin receptor isadministered IM or IV in doses of approximately 0.01 to about 1 mg/kg.

Interleukin receptor is administered, and then, about 12 hours to about96 hours after this, INFγ interferon is administered IM in doses ofabout 1 to about 3×10⁶ IU/kg body weight.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

1-99. (Canceled)
 100. A method of treating an allergy in a mammal, themethod comprising administering to said mammal an effective amount of apharmaceutical composition comprising a combination of an IgE inhibitorand a histamine inhibitor.
 101. The method of claim 100, wherein saidIgE inhibitor is an anti-IgE antibody, an anti-IgE receptor antibody ora soluble IgE receptor.
 102. The method of claim 100, wherein saidhistamine inhibitor is an anti-histamine antibody, an anti-histaminereceptor antibody or a soluble histamine receptor.
 103. The method ofclaim 100, wherein said method further comprises administering gammainterferon.
 104. The method of claim 100, wherein said mammal is ahuman.
 105. A kit for treating an allergy in a mammal, said kitcomprising an IgE inhibitor and a histamine inhibitor, an applicator andan instruction material for the use thereof.
 106. The kit of claim 105,wherein said IgE inhibitor is an anti-IgE antibody, an anti-IgE receptorantibody or a soluble IgE receptor.
 107. The kit of claim 105, whereinsaid histamine inhibitor is an anti-histamine antibody, ananti-histamine receptor antibody or a soluble histamine receptor.